Analogues of magainin peptides containing D-amino acids

ABSTRACT

Biologically active analogues of magainin peptides wherein each amino acid residue of the peptide is a D-amino acid residue or a glycine residue. Examples of such peptides wherein each amino acid residue is a D-amino acid residue or a glycine residue include deletion and substitution analogues of magainin peptides. Such peptides have increased resistance to proteolytic enzymes while retaining biological activity.

This application is a continuation, of application Ser. No. 08/133,740 filed Oct. 5, 1993, now abandoned; which is a continuation-in-part of application Ser. No. 07/874,685, filed Apr. 28, 1992, now abandoned; which is a continuation-in-part of application Ser. No. 07/522,688, filed May 14, 1990, now abandoned which is a continuation-in-part of application Serial No. 07/476,629 filed Feb. 8, 1990, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to biologically active peptides, and more particularly to biologically active peptides wherein each amino acid residue of such peptides is a D-aimino acid residue or glycine.

SUMMARY OF THE INVENTION

In accordance with an aspect of the present invention, there is provided compound comprising an analogue of Magainin I Peptide or Magainin II peptide. Each amino acid residue of the Magainin I peptide and the Magainin II peptide is a D-amino acid residue or glycine. The Magainin I or Magainin II peptide is in an amide or carboxy terminated form. Magainin I is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residues refer to the position of the residue in the peptide. ##STR1## Magainin II is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR2## The Magainin I or Magainin II peptide is substituted in at least one of positions 1-23. The substituents which may be employed in each of positions 1-23 are shown in the following table:

    ______________________________________                                         Residue No.                                                                            Substituent                                                            ______________________________________                                         1       D-Lys, D-Ala                                                           2       D-Lys, D-Ala, D-Ile, D-Arg, D-Leu, D-Val, D-His, D-Met                 3       D-Lys, D-Ala, D-Trp, D-Arg, D-His                                      4       D-Lys, D-Ala, D-Arg, D-His                                             5       D-Phe, D-Ala, D-Lys, D-Trp, D-Leu, D-Ile, D-Val, D-Met                 6       D-Leu, D-Lys, D-A1a, D-Ile, D-Val , D-Met                              7       D-Lys, D-His, D-Ala, D-Arg, D-Ile, D-Val, D-Met                        8       D-Ala, D-Lys, D-Ser, D-Trp, D-Met, D-Ile, D-Arg, D-His,                        D-Thr, D-Leu, D-Val                                                    9       D-Lys, D-Ala, D-Trp, D-Arg, D-His, D-Leu, D-Ile, D-Val                 10      D-Lys, D-Ala, D-Trp, D-Arg, D-His , D-Leu, D-Ile , D-Val               11      D-Lys, D-Arg, D-His                                                    12      D-Phe, D-Lys, D-Trp, D-Arg, D-His                                      13      D-Ala, D-Lys, D-Trp, D-Met, D-Arg, D-His, D-Phe,                               D-Leu, D-Ile, D-Val                                                    14      D-Ala, D-Lys, D-Arg, D-His                                             15      D-Lys, D-Trp, D-Ala, D-Arg, D-His, D-Phe                               16      D-Ala, D-Lys, D-Phe, D-Ile, D-Val, D-Met, D-Leu                        17      D-Ala, D-Lys, D-Val, D-Trp, D-Arg, D-His, D-Met, D-Leu                 18      D-Ala, D-Lys, D-Trp, D-Arg, D-His, D-Leu, D-Met                        19      D-Ala, D-Lys, D-Glu, Gly, D-Arg, D-His, D-Leu                                  D-Ile, D-Phe, D-Asn                                                    20      D-Ile, D-Ala, D-Lys, D-Trp, D-Leu, D-Phe, D-Val, D-Met                 21      D-Lys, D-Pro, D-Ala, D-His, D-Leu, D-Arg, D-Ile, D-Phe                 22      D-Lys, D-Ala, D-Asn, D-Gln, D-Arg, D-His                               23      D-Ser, D-Lys, D-Ala, D-Thr, Gly, D-Leu, D-Ile,                                 D-Gln, D-Asn                                                           ______________________________________                                    

Preferably, the substituents are as follows:

    ______________________________________                                         Residue No.     Substituent                                                    ______________________________________                                         1               D-Lys, D-Ala                                                   2               D-Lys, D-Ala, D-Ile                                            3               D-Lys, D-Ala, D-Trp                                            4               D-Lys, D-Ala                                                   5               D-Phe, D-Ala, D-Lys, D-Trp                                     6               D-Leu, D-Lys, D-Ala                                            7               D-Lys, D-His, D-Ala                                            8               D-Ala, D-Lys, D-Ser, D-Trp                                     9               D-Lys, D-Ala, D-Trp                                            10              D-Lys, D-Ala, D-Trp                                            11              D-Lys                                                          12              D-Phe, D-Lys, D-Trp                                            13              D-Ala, D-Lys, D-Trp                                            14              D-Ala, D-Lys                                                   15              D-Lys, D-Trp, D-Ala                                            16              D-Ala, D-Lys, D-Phe                                            17              D-Ala, D-Lys, D-Val, D-Trp                                     18              D-Ala, D-Lys, D-Trp                                            19              D-Ala, D-Lys, D-Glu                                            20              D-Ile, D-Ala, D-Lys, D-Trp                                     21              D-Lys, D-Pro, D-Ala                                            22              D-Lys, D-Ala, D-Asn                                            23              D-Ser, D-Lys, D-Ala                                            ______________________________________                                    

It is to be understood that for purposes of the present invention, that the D-tryptophan residues may be protected with a formyl group or be unprotected. The D-phenylalanine residues, whether such residue is present in its normal position, or employed as a substitution residue, may be a normal D-phenylalanine residue or an iodinated D-phenylalanine residue.

DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with one embodiment, the peptide is a Magainin I peptide, and at least one of amino acid residues 8, 10, 13, 16, 18 and 19 is substituted with a D-alanine residue. In another embodiment, the Magainin I peptide has D-alanine residue substitutions at each of amino acid residues 3, 8, 16, 19 and 23.

In accordance with another embodiment, the Magainin I peptide has D-alanine residue substitutions at four of amino acid residues 3, 8, 16, 19 and 23, and the remaining one of these residues is substituted with a D-lysine residue

In accordance with yet a further embodiment, amino acid 21 of a Magainin I peptide is substituted with a D-proline residue.

In accordance with another embodiment, at least one of amino acid residues 3, 7, 8, 10, 18-21, and 23 of Magainin I is substituted with a D-lysine residue.

In accordance with another embodiment, amino acid residues 3, 8, 9, 19 and 23 of Magainin I are each substituted with a D-lysine residue and amino acid residue 16 is substituted with a D-alanine residue.

In accordance with a further embodiment, at least one of amino acid residues 3, 5, 8, 10, 12, 13, 15, 18, and 20 of Magainin I is substituted with a protected D-tryptophan residue, wherein the protecting group is preferably a formyl group. In accordance with another embodiment, at least one of amino acid residues 9, 13, 15 and 17 of Magainin I is substituted with an unprotected D-tryptophan residue.

In accordance with another embodiment, the peptide is a Magainin II peptide, and at least one of amino acid residues 1-8, 10, 13, 14, 16 and 18-23 is a D-alanine residue.

In accordance with another embodiment, at least one of amino acid residues 1-3, 5-9, 12, 13 and 15-23 of Magainin II is a D-lysine residue.

In accordance with another aspect of the present invention, there is provided an analogue of Magainin I or Magainin II peptide, said Magainin I or Magainin II peptide being in an amide- or carboxy-terminated form and having the structural formulas hereinabove described, wherein each amino acid residue of said Magainin I or Magainin II peptide is a D-amino acid residue or glycine, and wherein at least one of amino acid residues 15-23 is omitted and at least one of the remaining amino acid residues is substituted. The substituents which may be employed in at least one of positions 1-23 are shown in the following table:

    ______________________________________                                         Residue No.                                                                            Substituent                                                            ______________________________________                                         1       D-Lys, D-Ala                                                           2       D-Lys, D-Ala, D-Ile, D-Arg, D-Leu,                                             D-Val, D-His, D-Met                                                    3       D-Lys, D-Ala, D-Trp, D-Arg, D-His                                      4       D-Lys, D-Ala, D-Arg, D-His                                             5       D-Phe, D-Ala, D-Lys, D-Trp, D-Leu, D-Ile, D-Val, D-Met                 6       D-Leu, D-Lys, D-Ala, D-Ile, D-Val, D-Met                               7       D-Lys, D-His, D-Ala, D-Arg, D-Ile, D-Val, D-Met                        8       D-Ala, D-Lys, D-Ser, D-Trp, D-Met, D-Ile, D-Arg,                               D-His, D-Thr, D-Leu, D-Val                                             9       D-Lys, D-Ala, D-Trp, D-Arg, D-His, D-Leu, D-Ile, D-Val                 10      D-Lys, D-Ala, D-Trp, D-Arg, D-His, D-Leu, D-Ile, D-Val                 11      D-Lys, D-Arg, D-His                                                    12      D-Phe, D-Lys, D-Trp, D-Arg, D-His                                      13      D-Ala, D-Lys, D-Trp, D-Met, D-Arg, D-His, D-Phe,                               D-Leu, D-Ile, D-Val                                                    14      D-Ala, D-Lys, D-Arg, D-His                                             15      D-Lys, D-Trp, D-Ala, D-Arg, D-His, D-Phe                               16      D-Ala, D-Lys, D-Phe, D-Ile, D-Val, D-Met, D-Leu                        17      D-Ala, D-Lys, D-Val, D-Trp, D-Arg, D-His, D-Met, D-Leu                 18      D-Ala, D-Lys, D-Trp, D-Arg, D-His, D-Leu, D-Met, D-Phe                 19      D-Ala, D-Lys, D-Glu, Gly, D-Arg, D-His, D-Leu,                                 D-Ile, D-Phe, D-Asn                                                    20      D-Ile, D-Ala, D-Lys, D-Trp, D-Leu, D-Phe, D-Val, D-Met                 21      D-Lys, D-Pro, D-Ala, D-His, D-Leu, D-Arg, D-Ile, D-Phe                 22      D-Lys, D-Ala, D-Asn, D-Gln, D-Arg, D-His                               23      D-Ser, D-Lys, D-Ala, D-Thr, Gly, D-Leu, D-Ile,                                 D-Gln, D-Asn                                                           ______________________________________                                    

Preferably, the substituents are as follows:

    ______________________________________                                         Residue No.     Substituent                                                    ______________________________________                                         1               D-Lys, D-Ala                                                   2               D-Lys, D-Ala, D-Ile                                            3               D-Lys, D-Ala, D-Trp                                            4               D-Lys, D-Ala                                                   5               D-Phe, D-Ala, D-Lys, D-Trp                                     6               D-Leu, D-Lys, D-Ala                                            7               D-Lys, D-His, D-Ala                                            8               D-Ala, D-Lys, D-Ser, D-Trp                                     9               D-Lys, D-Ala, D-Trp                                            10              D-Lys, D-Ala, D-Trp                                            11              D-Lys                                                          12              D-Phe, D-Lys, D-Trp                                            13              D-Ala, D-Lys, D-Trp                                            14              D-Ala, D-Lys                                                   15              D-Lys, D-Trp, D-Ala                                            16              D-Ala, D-Lys, D-Phe                                            17              D-Ala, D-Lys, D-Val, D-Trp                                     18              D-Ala, D-Lys, D-Trp                                            19              D-Ala, D-Lys, D-Glu                                            20              D-Ile, D-Ala, D-Lys, D-Trp                                     21              D-Lys, D-Pro, D-Ala                                            22              D-Lys, D-Ala, D-Asn                                            23              D-Ser, D-lys, D-Ala                                            ______________________________________                                    

In accordance with a particular embodiment, amino acid residues 16-23 of Magainin I are deleted, and amino acid residues 3, 8 and 10 are substituted with a D-alanine residue.

In accordance with yet another embodiment, amino acid residue 19 of Magainin I is deleted, and preferably amino acid residues 5, 8, 9 and 16 are each substituted with a D-lysine residue, amino acid residue 21 is substituted with a D-leucine residue, and amino acid residues 18 and 23 are substituted with a D-alanine residue.

In accordance with another embodiment, in carboxy- or amide-terminated (preferably amide-terminated) Magainin I or Magainin II peptide, amino acid residues 17-23 or 16-23 or 15-23 are deleted, and amino acid residues 3, 7, and 8 are each substituted with a D-lysine residue, and optionally amino acid residue 13 and/or amino acid residue 10 is substituted with a D-alanine residue. Preferred peptides are as follows:

GIKKFLKKAGKFGK-NH₂

GIKKFLKKAGKFGKAF-NH₂

GIKKFLKKAKKFGKA-NH₂

GIKKFLKKAKKFAKA-NH₂

GIKKFLKKAAKFAKA-NH₂

Preliminary studies indicate that the above-mentioned preferred peptides possess low hemolytic activity. In accordance with a further embodiment, amino acid residue 21 of Magainin I may be deleted, and preferably amino acid residues 5, 10, 18, and 19 are each substituted with a D-lysine residue, amino acid residue 7 is substituted with a D-phenylalanine residue, and amino acid residue 22 is substituted with a D-alanine residue.

In accordance with another embodiment, in carboxy or amide terminated (preferably amide terminated) Magainin I or Magainin II (perferably Magainin II), amino acid residue 19 is omitted, and at least one of amino acid residues 3, 7, 8, 10, 13, 15, 16, 18 21, 22 or 23 is substituted with another amino acid as follows:

    ______________________________________                                         Residue number  Subtituent                                                     ______________________________________                                         3               D-Leu                                                          7               D-Lys                                                          8               D-Lys, D-Ala                                                   10              D-Ala, D-Lys                                                   13              D-Trp, D-Leu, D-Phe, D-Ala                                     15              D-Phe                                                          16              D-Ala                                                          18              D-Lys, D-Ala, D-Phe                                            21              D-Lys, D-Ile                                                   22              D-Lys                                                          23              D-Lys, D-Ser                                                   ______________________________________                                    

In a preferred embodiment, the peptide is a Magainin II peptide, and the substitution analogue wherein amino acid 19 is deleted is selected from the class consisting of the following substitution analogues:

GIGKFLKKAKKFGKAFVKILKK;

GIGKFLKSAKKFGKAFVKIMNS;

GIGKFLKKAKKFGKAFVKIMKK;

GIGKFLKSAKKFGKAFVKILNS;

GIGKFLKKAKKFAKAFVKIINN;

GIGKFLHSAKKFGKAFVGIMKS;

GIGKFLHSAKKFGKAFVAIMKS;

GIGKFLHSAKKFFKAFVFIMNS;

GIGKFLKSAKKFGKAFVFIMNS;

GIGKFLHKAKKFAKAFVFIMNS;

GIGKFLKSAKKFAKAFVFIMNS;

GIGKFLHKAKKFAKAFVFIMNK;

GIGKFLKKAKKFGKAFVFIMKK; and

GIGKFLHSAKKFGKAFVK**IMNS; wherein

K** is E-F-moc-lysine.

Preliminary studies indicate that these preferred analogues possess low hemolytic activity.

In accordance with another embodiment, there are provided derivatives of Magainin I or Magainin II (carboxy- or amide-terminated), preferably Magainin II, wherein a portion of the basic peptide is deleted and at least one of the remaining amino acid residues is substituted as hereinabove described; in particular, amino acid residue 19 is omitted and in addition either amino acid residues 1-4 or 3,5, and 6 are omitted. Preferred peptides are as follows:

FLHSAKKFGKAFVFIMNS

GIKHSAKKFAKAFKAIMNS

FLKSAKKFGKAFVGIMNS

FLKSAKKFAKAFVGIMNS

In accordance with another aspect of the present invention, there is provided a peptide which is a deletion analogue of an amide or carboxy terminated Magainin I, wherein Magainin I is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR3## and wherein each amino acid residue is a D-amino acid residue or a glycine residue, and at least one of amino acid residues 15 through 23 is omitted. In a preferred embodiment, at least one of amino acid residues 15, 16, 18, 19, 21, 22 and 23 is omitted. In one embodiment, at least amino acid residue 18 is omitted whereas in another embodiment, at least amino acid residue 19 is omitted, and in yet another embodiment at least amino acid residue 21 is omitted. In preferred embodiments, only one of amino acid residues 18, 19, and 21, respectively, is omitted. In other preferred embodiments, amino acid residues 21, 22, and 23 are omitted, amino acid residues 19 through 23 are omitted, amino acid residues 18 through 23 are omitted, and amino acid residues 17 through 23 are omitted. The compound can be a deletion analogue of amide--or carboxy-terminated Magainin I.

In accordance with another aspect of the present invention, there is provided a peptide which is a deletion analogue of an amide--or carboxy-terminated Magainin II, wherein Magainin II is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR4## wherein each amino acid residue is a D-amino acid residue or a glycine residue, and at least one of amino acids 15 through 22 is omitted. In a preferred embodiment, at least one of amino acids 15, 18, 19, 20, 21, and 22 is omitted. In one embodiment, at least amino acid 18 is omitted. In another embodiment, at least amino acid 19 is omitted. Another embodiment omits at least amino acid 21, and yet other embodiment omits at least amino acid 22. In preferred embodiments, only one of amino acids 18, 19, 21, and 22, respectively, is omitted. The compound can be a deletion analogue of amide-terminated Magainin II.

In accordance with yet another aspect of the present invention, there is provided a basic (positively charged) polypeptide having at least sixteen amino acids wherein the polypeptide includes at least eight hydrophobic amino acids and at least eight hydrophilic amino acids. Each of the amino acid residues of the polypeptide is a D-amino acid residue or a glycine residue. Still more particularly, the hydrophobic amino acids are in groups of two adjacent amino acids wherein the amino acids are D-amino acids or glycine, and each group of two hydrophobic amino acids is spaced from another group of two hydrophobic amino acids by at least one D-amino acid other than a hydrophobic amino acid (preferably at least two D-amino acids) and generally by no greater than four D-amino acids, and the D-amino acids between pairs of hydrophobic amino acids may or may not be hydrophilic.

The hydrophilic amino acids are generally also in groups of two adjacent D-amino acids in which at least one of the two D-amino acids is a basic hydrophilic amino acid, with such groups of two hydrophilic D-amino acids being spaced from each other by at least one amino acid, other than a hydrophilic D-amino acid, wherein each of said at least one amino acid(s) is a D-amino acid or glycine (preferably at least two amino acids) and generally no greater than four amino acids, and the amino acids between pairs of hydrophilic D-amino acids may or may not be hydrophobic.

In accordance with a particularly preferred embodiment, the polypeptide comprises a chain of at least four groups of amino acids wherein each amino acid is a D-amino acid or glycine, with each group consisting of four amino acids wherein each of the at least four amino acids is a D-amino acid or glycine. Two of the four amino acids in each group are hydrophobic amino acids, and two of the four amino acids in each group are hydrophilic, with at least one of the hydrophilic amino acids in each group being a basic hydrophilic amino acid and the other being a basic or neutral hydrophilic amino acid.

The hydrophobic amino acids may be selected from the class consisting of D-Ala, D-Cys, D-Phe, D-Ile, D-Leu, D-Met, D-Val, D-Trp, D-Tyr, and Gly. The neutral hydrophilic D-amino acids may be selected from the class consisting of D-Ser, D-Asn, D-Gln, and D-Thr. The basic hydrophilic D-amino acids may be selected from the class consisting of D-Lys, D-Arg, and D-His, D-ornithine (D-Orn), D-homoarginine (D-Har), D-2,4-diaminobutyric acid (D-Dbu), and D-p-aminophenylalanine.

Each of the groups of four amino acids may be of the sequence ABCD, BCDA, CDAB, or DABC, wherein A and B are each hydrophobic amino acids and may be the same or different, one of C or D is a basic hydrophilic amino acid, and the other of C or D is a basic or neutral hydrophilic amino acid and may be the same or different. In a preferred embodiment, the polypeptide chain may comprise 5 or 6 groups of this sequence. In each group, each of A, B, C and D may be the same in some or all of the groups or may be different in some or all of the groups.

The polypeptide chain preferably has at least 20 amino acids, and no greater than 50 amino acids wherein each amino acid is a D-amino acid or glycine. It is to be understood, however, that the polypeptide does not have to consist entirely of the groups described above. The polypeptide may have amino acids extending from either or both ends of the noted groups forming the polypeptide chain and/or there may be amino acids between one or more of the at least four groups and still remain within the scope of the invention, provided that each amino acid residue of the polypeptide chain is a D-amino acid residue or glycine.

The groups of amino acids may be repeating groups of amino acids, or the amino acids in the various groups may vary provided that in each group of the at least four groups of amino acids there are two hydrophobic amino acids wherein each amino acid is a D-amino acid or glycine, and two hydrophilic D-amino acids as hereinabove noted.

Thus, in a preferred embodiment, the biologically active polypeptide comprises a chain including at least four groups of amino acids, each containing four amino acids wherein each amino acid is a D-amino acid or glycine. Two of the four amino acids in each group are hydrophobic, wherein each hydrophobic amino acid is a D-amino acid or glycine, at least one amino acid is a basic hydrophilic D-amino acid, and the remaining one is a basic or neutral hydrophilic D-amino acid, with the polypeptide chain preferably having at least 20 amino acids but no greater than 50 amino acids.

In one embodiment, each of the at least four groups of amino acids which are in the peptide chain is of the sequence A-B-C-D, B-C-D-A, C-D-A-B or D-A-B-C wherein A and B are hydrophobic, one of C or D is basic hydrophilic, and the other of C or D is basic or neutral hydrophilic. The resulting polypeptide chain, therefore, may have one of the following sequences:

(X₁)_(a) (A-B-C-D )_(n) (Y₁)

(X₂)(B-C-D-A)_(n) (Y₂)_(b)

(X₃)_(a) (C-D-A-B)_(n) (Y₃)_(b)

(X₄)_(a) (D-A-B-C)_(n) (Y₄)_(b)

wherein X₁ is D; C-D- or B-C-D-, Y₁ is -A or -A--B or -A-B-C

X₂ is A-, D-A- or C-D-A-

Y₂ is -B, -B-C or B-C-D

X₃ is B-, A-B-, D-A-B-

Y₃ is -C, -C-D, -C-D-A

X₄ is C-, B-C-, A-B-C-

Y₄ is -D, -D-A, -D-A-B

a is 0 or 1; b is 0 or 1

and n is at least 4.

It is to be understood that the peptide chain may include D-amino acids or glycine between the hereinabove noted groups of four amino acids provided that the spacing between such groups and the charge on the D-amino acids or glycine does not change the characteristics of the peptide chain which provide amphiphilicity and a positive charge and do not adversely affect the folding characteristics of the chain to that which is significantly different from one in which the hereinabove noted groups of four amino acids are not spaced from each other.

As representative examples, there may be mentioned. ##STR5##

The peptide may have amino acids extending from either end of the chain. For example, the chains may have a D-Ser-D-Lys sequence before the "D-Ala" end, and/or a D-Ala-D-Phe sequence after the "D-Lys" end. Other amino acid sequences may also be attached to the "D-Ala" and/or the "D-Lys" end.

Similarly, in any polypeptide chain of the present invention having at least four groups of amino acids of the sequence as described above, the chain may have, for example, a C-D sequence before the first A-B-C-D group. Also other acid sequences including D-amino acids or glycine may be attached to the "A" and/or the "D" end of one of these polypeptide chains. Also there may be D-amino acids or glycine residues in the chain which space one or more groups of the hereinabove noted four amino acids from each other.

Such polypeptides are generally water-soluble to a concentration of at least 20 mg/ml at neutral pH in water. Such polypeptides are non-hemolytic, i.e., they will not rupture red blood cells at effective antimicrobial concentrations. The structure of such polypeptides provides for flexibility of the polypeptide molecule. When the polypeptide is placed in water, it does not assume an amphiphilic structure. When the polypeptide encounters an oily surface or membrane, the polypeptide chain folds upon itself into a rod like structure.

In accordance with a further aspect of the present invention, there is provided a peptide (polypeptide) having from eight to fifteen amino acids comprised of at least four hydrophobic amino acids and four hydrophilic amino acids. Each amino acid residue is a D-amino acid residue or glycine. The hydrophobic amino acids are in groups of two adjacent hydrophobic D-amino acids or glycine wherein each group of two hydrophobic D-amino acids or glycine is spaced from each other by at least one D-amino acid other than a hydrophobic D-amino acid or glycine (preferably at least two D-amino acids) and generally no greater than four D-amino acids, and the D-amino acid(s) between pairs of hydrophobic amino acids wherein each amino acid is a D-amino acid or glycine, may or may not be hydrophilic. The hydrophilic D-amino acids are generally also in groups of two adjacent D-amino acids in which at least one of the two D-amino acids is a basic hydrophilic D-amino acid and the other of the two is basic or neutral. The groups of two hydrophilic D-amino acids are spaced from each other by at least one amino acid other than a hydrophilic D-amino acid, and wherein the at least one amino acid is a D-amino acid or glycine (preferably at least two amino acids) and generally no greater than 4 amino acids, and the amino acids between pairs of hydrophilic amino acids may or may not be hydrophobic.

The peptide having from 8 to 15 amino acids is amphiphilic and is positively charged (basic).

The 8 to 15 amino acid peptide hereinabove described may or may not be bioactive, and in the case where such peptide is not bioactive, it has utility as an intermediate in providing the hereinabove noted peptides which have at least 16 amino acids and which are bioactive. For example, two peptides, one having eight D-amino acids and the other having twelve D-amino acids may be coupled to each other to form a peptide having 20 D-amino acids of the type hereinabove described and which is bioactive. The peptides may be coupled by standard peptide chemistry techniques. Thus, for example, such peptides may be condensed in solution by the technique disclosed by Johnson, et al. Peptides, pages 239-42 (Walter de Gruzter & Co., 1986).

As representative examples of such peptides, there may be mentioned peptides represented by the following structure wherein A, B, C and D are as defined previously;

(i) (W₁)_(a) (A-B-C-D)_(n) (Z₁)_(b)

(ii) (W₂)_(a) (B-C-D-A)_(n) (Z₂)_(b)

(iii) (W₃)_(a) (C-D-A-B)_(n) (Z₃)_(b)

(IV) (W₄)_(a) (D-A-B-C)_(n) (Z₄)_(b)

wherein W₁ is D-, C-D-, B-C-D-

W₂ is A-, D-A-, C-D-A-

W₃ is B-, A-B-, D-A-B-

W₄ is C-, B-C-, A-B-C-

Z₁ is -A, -A-B, -A-B-C

Z₂ is -B, -B-C, -B-C-D

Z₃ is -C, -C-D, -C-D-A

Z₄ is -D, -D-A, -D-A-B

n is 2 or 3, a is 0 or 1 and b is 0 or 1.

In accordance with yet another aspect of the present invention, there is provided a peptide which includes the following basic peptide structure X:

-R₁ -R₁ -R₂ -R₂ -R₁ -R₁ -R₃ -R₁ -

-R₁ -R₁ -R₃ -R₁ -R₁ -R₄ -R₅ -R₁ -

wherein R₁ is a hydrophobic amino acid;

R₂ is a hydrophobic amino acid or a basic hydrophilic amino acid;

R₃ is a basic hydrophilic amino acid;

R₄ is a hydrophobic or neutral hydrophilic amino acid;

R₅ is a basic or neutral hydrophilic amino acid, and each amino acid residue of said peptide is a D-amino acid residue or glycine.

The hereinabove basic structure is hereinafter symbolically indicated as X. Peptides formed in accordance with this basic structure are commonly referred to as CPF peptides.

The CPF peptides of the present invention may include only the hereinabove noted D-amino acids or glycine or may include additional D-amino acids or glycine residues at the amino and/or carboxyl end or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids, wherein each amino acid is a D-amino acid or glycine.

The CPF peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end, wherein each amino acid is a D-amino acid or glycine.

Accordingly, such preferred peptides may be represented by the structural formula:

    Y--X--

wherein X is the hereinabove described basic peptide structure and Y is

(i) R₅ -, or

(ii) R₂ -R₅ -; or

(iii) R₁ -R₂ -R₅ ; or

(iv) R₂ -R₁ -R₂ -R₅ ; preferably Glycine -R₁ -R₂ -R₅.

wherein R₁, R₂ and R₅ are as previously defined.

The carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.

In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:

-X -Z wherein

X is the hereinabove defined basic peptide structure and Z is

(i) R₁ -, or

(ii) R₁ -R₁ -; or

(iii) R₁ -R₁ -R₄ ; or

(iv) R₁ R₁ -R₄ -R₄ ; or

(v) R₁ -R₁ -R₄ -R₄ -R₆ ; or

(vi) R₁ -R₁ -R₄ -R₄ -R₆ -D-Gln; or

(vii) R₁ -R₁ -R₄ -R₄ -R₆ -D-Gln-D-Gln, wherein R₁ and R₄ are as previously defined, and R6 is D-proline or a hydrophobic amino acid.

Preferred peptides may be represented by the following structural formula

    (Y).sub.a -X-(Z).sub.b

wherein X, Y and Z are as previously defined and a is 0 or 1 and b is 0 or 1.

As representative examples of CPF peptides used in the present invention, there may be mentioned peptides represented by the following (single letter amino acid code):

G12S3LG4ALKA5LKIG678LGG9(10)QQ

Where:

1=F, L

2=G, A

3=F, L

4=K, L

5=A, G, T

6=A, T

7=H, N

8=A, M, F, L

9=A, S, T

10=P, L

Preferred CPF peptides are of the following sequences:

(1) GFGSFLGLALKAALKIGANALGGAPQQ

(2) GLASFLGKALKAGLKIGAHLLGGAPQQ

(3) GLASLLGKALKAGLKIGTHFLGGAPQQ

(4) GLASLLGKALKATLKIGTHFLGGAPQQ

(5) GFASFLGKALKAALKIGANMLGGTPQQ

(6) GFGSFLGKALKAALKIGANALGGAPQQ

(7) GFGSFLGKALKAALKIGANALGGSPQQ

(8) GFASFLGKALKAALKIGANLLGGTPQQ

In accordance with another aspect of the present invention, there is provided a composition comprising at least one biologically active peptide which includes at least a chain of the following 20 amino acid residues having the following peptide structure X₁₀ where X₁₀ is:

-R₁₁ -R₁₂ -R₁₁ -R₁₁ -R₁₃ -R₁₃ - R₁₁ -R₁₁ -R₁₂ -R₁₁ -R₁₁ -R₁₅ -R₁₄ -R₁₁ -R₁₂ -R₁₃ -

-R₁₁ -R₁₆ -R₁₅ -R₁₇, wherein:

R₁₁ is a hydrophobic amino acid;

R₁₂ is a basic hydrophilic amino acid;

R₁₃ is a hydrophobic or basic: hydrophilic amino acid;

R₁₄ is a netural hydrophilic amino acid, or a basic hydrophilic amino acid;

R₁₅ is a neutral hydrophilic amino acid; R₁₆ is D-proline or a hydrophobic amino acid; and R₁₇ is a netural hydrophilic, basic hydrophilic or hydrophobic amino acid. Each of the amino acid residues of the peptide is a D-amino acid residue or glycine. The at least one biologically active peptide which includes the peptide structure X₁₀ has at least the hereinabove described twenty amino acid peptide and generally no greater than 35 amino acids, preferably no greater than 30 amino acids. Such peptides are derived from fragments of the human hormone cholecystokinin, or may be derivatives of such fragments.

In one embodiment, the at least one biologically active peptide is of the formula R₁₁ -R₁₁ -X-,

wherein R₁₁ is a hydrophobic amino acid as described above. Most preferably, the biologically active peptide of this embodiment includes a chain of at least the following amino acids:

D-Leucine-D-Leucine-X-.

In another embodiment, the at least one biologically active petpide includes a chain of at least the following amino acids:

-X-R₁₂ -R₁₁ -R₁₅ -R₁₁ -R₁₁ -, wherein R₁₁ R₁₂, and R₁₅ are amino acids of the types hereinabove described.

The peptide may be amide-terminated or carboxy-terminated.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₁₂ :

R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂ !_(n'), wherein R₂₁ is a basic hydrophilic amino acid, R₂₂ is a hydrophobic amino acid, R₂₃ is a neutral hydrophilic or hydrophobic amino acid, n is from 2 to 5, and each amino acid residue of the peptide is a D-amino acid. residue or glycine.

In accordance with one embodiment, the peptide may include the following structure:

Y₁₂ -X₁₂, wherein X₁₂ is as hereinabove described, and Y is:

(i) R₂₂ ;

(ii) R₂₂ -R₂₂ ;

(iii) R₂₁ -R₂₂ -R₂₂ ;

(iv) R₂₃ -R₂₁ -R₂₂ R₂₂ ;

(v) R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂ ; or

(vi) R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂, wherein R₂₁, R₂₂, and R₂₃ are as hereinabove described

In accordance with another embodiment, the peptide may include the following structure:

X₁₂ -Z₁₂, wherein X₁₂ is as hereinabove described, and Z₁₂ is:

(i) R₂₁ ;

(ii) R₂₁ -R₂₂ ;

(iii) R₂₁ -R₂₂ -R₂₂

(iv) R₂₁ -R₂₂ -R₂₂ -R₂₃ ;

(v) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ ; or

(vi) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂.

In accordance with yet another embodiment, the peptide may include the following structure:

(Y₁₂)_(a) -X₁₂ -(Z₁₂)_(b), wherein Y and Z are as previously defined, a is 0 or 1, and b is 0 or 1.

In a preferred embodiment, n is 3, and most preferably the peptide has the following structure as indicated by the single letter amino acid code:

KIAGKIA!₃.

In another embodiment, n is 3, and the peptide has the following structural formula:

Lys Ile Ala Gly (p-aminophenylalanine) Ile Ala!₃

In another embodiment, n is 2, and the peptide preferably is of the following structure as indicated by the single letter amino acid code:

KIA(KIAGKIA)₂ KIAG.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₁₄ :

R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂ -R₂₁ -R₂₂ -R₂₂ -R₂₂ -R₂₁ -R₂₂ -R₂₂,

wherein R₂₁, R₂₂ and R₂₃ are as hereinabove described, and each amino acid residue of the peptide is a D-amino acid residue or glycine.

In accordance with one embodiment, the peptide may include the following structure:

Y₁₄ -X₁₄, wherein X₁₄ is as hereinabove described, and Y14 is:

(i) R₂₂ ;

(ii) R₂₂ -R₂₂ ;

(iii) R₂₁ -R₂₂ -R₂₂ ;

(iv) R₂₃ -R₂₁ -R₂₂ -R₂₂ ;

(v) R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂ ;

(vi) R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂, or

(vii) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂, wherein R₂₁, R₂₂ and R₂₃ are as hereinabove described.

In accordance with another embodiment, the peptide may include the following structure:

X₁₄ -Z₁₄, wherein X₁₄ is as hereinabove described and Z₁₄ is:

(i) R₂₁ ;

(ii) R₂₁ -R₂₂ ;

(iii) R₂₁ -R₂₂ -R₂₂ ;

(iv) R₂₁ -R₂₂ -R₂₂ -R₂₃ ;

(v) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ ;

(vi) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ ; or

(vii) R₂₁ -R₂₂ -R₂₂ -R₂₃ -R₂₁ -R₂₂ -R₂₂, wherein R₂₁, R₂₂ and R₂₃ are as hereinabove described.

In accordance with yet another embodiment the peptide may include the following structure:

(Y₁₄)a-X₁₄ -(Z₁₄)b, wherein X₁₄, Y₁₄ and Z₁₄ are as previously defined, a is 0 or 1, and b is 0 or 1. In a preferred embodiment, the peptide is of the following structural formula as indicated by the single letter amino acid code:

KLASKAGKIAGKIAKVALKAL.

In another preferred embodiment, the peptide is of the following structural formula as indicated by the single letter amino acid code:

KIAGKIAKIAGOIAKIAGKIA.

In accordance with a further aspect of the present invention, the peptide may include the following basic peptide structure X₁₆ :

-R₃₁ -R₃₃ -R₃₂ -R₃₁ -R₃₄ -R₃₄ -R₃₁ -

-R₃₁ -R₃₄ -R₃₂ -R₃₁ -R₃₁ -R₃₂ -R₃₁ -

-R₃₁ -R₃₁ -R₃₂ -

wherein R₃₁ is a hydrophobic amino acid, R₃₂ is a basic hydrophilic amino acid, R₃₃ is a neutral hydrophilic, basic hydrophilic, or hydrophobic amino acid, and R₃₄ is a hydrophobic or basic hydrophilic amino acid, and each amino acid residue is a D-amino acid residue or glycine. Preferably, R₃₃ is a neutral hydrophilic amino acid.

Such peptides are commonly referred to as PGLa peptides.

The PGLa peptides generally include at least seventeen amino acids and may include as many as forty amino acids. Accordingly, the hereinabove described basic peptide structure for a PGLa peptide may include additional amino acids at the amino end or at the carboxyl end or at both the amino and carboxyl end.

Thus, for example, a PGLa peptide may have the following structure:

-Y₁ -X₁₆

where X₁₆ is a previously defined; and Y16 is:

(i) R₃₁ ; or

(ii) R₃₄ -R₃₁

where R₃₁ and R₃₄ are as previously defined.

For example, a PGLa peptide may also have the following structure:

-X16-Z₁₆

where X₁₆ is a previously defined; and Z₁₆ is:

Y₁₆ is

(i) R₃₁ ; or

(ii) R₃₁ -R₃₁

where R₃₁ is as previously defined.

A PGLa peptide may also have the following structure:

(Y₁₆)a-X₁₆ -(Z₁₆)b

where X₁₆, Y₁₆, and Z₁₆ are as previously defined, a is 0 or 1 and b is 0 or 1.

In accordance with another embodiment, the peptide may include the following basic peptide structure X₁₈ :

-R₃₁ -R₃₃ -R₃₂ -R₃₁ -R₃₄ -R₃₅ -R₃₃ -

R₃₁ -R₃₄ -R₃₂ -R₃₁ -R₃₁ -R₃₂ -

R₃₁ -R₃₁ -R₃₁ -R₃₂ -(R₃₆)_(n) -R₃₁ -, wherein R₃₁, R₃₂, R₃₃, and R₃₄ are as previously defined, and R₃₅ is glutamine or asparagine or a basic hydrophilic, or hydrophobic amino acid, R₃₆ is glutamic acid, aspartic acid, a hydrophobic amino acid or a basic hydrophilic amino acid, and n is 0 or 1. Each amino acid residue is a D-amino acid residue or glycine.

Such peptides are commonly referred to as XPF peptides.

The XPF peptides generally include at least nineteen amino acids and may include up to forty amino acids. Accordingly, the hereinabove described basic peptide structure of XPF may include additional amino acids at the amino and carboxyl ends.

Thus, for example, an XPF peptide may include the following structure:

-Y₁₈ -X₁₈ -

where X₁₈ is as previously defined and Y₁₈ is

(i) R₃₁ or

(ii) R₃₄ -R₃₁

where R₃₁ and R₃₄ are as previously defined.

An XPF peptide may include the following structure:

-X₁₈ -Z₁₈ -

where X₁₈ is as previously defined and Z₁₈ is

(i) R₃₁ ; or

(ii) R₃₁ -R₃₅ ; or

(iii) R₃₁ -R₃₅ -D-Proline; or

(iv) R₃₁ -R₃₅ -D-Proline-R₃₂

An XPF peptide may also have the following structure;

(Y₁₈)a-X₁₈ (Z₁₈)b

where X₁₈, Y₁₈ and Z₁₈ are as previously defined, a is 0 or 1, and b is 0 or 1.

Preferred are XPF or PGLa peptides, which are characterized by the following primary amino acid sequence (single letter amino acid code):

PGLa: GMASKAGAIAGKIAKVALKAL (NH₂)

XPF: GWASKIGQTLGKIAKVGLKELIQPK

A review of XPF and PGLa can be found in Hoffmann et al., EMBO J. 2:711-714, 1983; Andreu et al., J.Biochem, 149:531-535, 1985; Gibson et al. J. Biol Chem. 261:5341-5349, 1986; and Giovannini et al, Biochem J. 243:113-120, 1987.

In accordance with another aspect of the present invention, there is provided an analogue of a biologically active amphiphilic amide or carboxy-terminated peptide, said peptide being represented by the following structural formula, and wherein the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR6##

R₁ is a hydrophobic amino acid, R₂ is a hydrophobic amino acid or a basic hydrophilic amino acid, R₃ is a basic hydrophilic amino acid, R₄ is a hydrophobic or neutral hydrophilic amino acid, R₅ is a basic hydrophilic or a neutral hydrophilic amino acid, and R₆ is a neutral hydrophilic amino acid. Each amino acid residue of the peptide is a D-amino acid residue or a glycine residue. At least one of and no more than seven of amino acid residues 2 through 26 are deleted from the peptide. In one embodiment, one of amino acid residues 2 through 26 is deleted from the peptide.

The hydrophobic amino acids are D-Ala, D-Cys, D-Phe, Gly, D-Ile, D-Leu, D-Met, D-Pro, D-Val, D-Trp, D-Tyr, D-norleucine (D-Nle), D-norvaline (D-Nval), and D-cyclohexylalanine (D-Cha).

The neutral hydrophilic amino acids are D-Asn, D-Gln, D-Ser, and D-Thr.

The basic hydrophilic amino acids are D-Lys, D-Arg, D-His, D-Orn, D-homoarginine (D-Har), D-2,4-diaminobutyric acid (D-Dbu), and D-p-aminophenylalanine.

Such analogues are sometimes hereinafter referred to as "deletion analogues." Representative examples of such deletion analogues comprise the following sequences:

(1) GASFLGKALKAALKIGANLLGGTPQQ

(2) GFSFLGKALKAALKIGANLLGGTPQQ

(3) GFAFLGKALKAALKIGANLLGGTPQQ

(4) GFASLGKALKAALKIGANLLGGTPQQ

(5) GFASFGKALKAALKIGANLLGGTPQQ

(6) GFASFLGKALKAALKGANLLGGTPQQ

(7) GFASFLGRALKAALKIANLLGGTPQQ

(8) GFASFLGKALKAALKIGNLLGGTPQQ

(9) GFASFLGKALKAALKIGALLGGTPQQ

(10) GFASFLGKALKAALKIGANLGGTPQQ

(11) GFASFLGKALKAALKIGANLLGTPQQ

(12) GFASFLGKALKAALKIGANLLGGPQQ

(13) GFASFLGKALKAALKIGANLLGGTQQ

(14) GFASFLGKALKAALKIGANLLGGTPQ

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula: ##STR7##

Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic peptide structure X₂₀ :

-R₃ -R₁ -R₂ -R₂ -R₁ -R₁ -R₃ -R₁ -

-R₁ -R₁ -R₃ -R₁ -R₁ -R₄ -R₅ -R₁ -

wherein R₁ is a hydrophobic amino acid;

R₂ is a hydrophobic amino acid or a basic hydrophilic amino acid;

R₃ is a basic hydrophilic amino acid;

R₄ is a hydrophobic or neutral hydrophilic amino acid; and

R₅ is a basic or neutral hydrophilic amino acid.

Each amino acid residue is a D-amino acid residue or a glycine residue.

The hereinabove basic structure is hereinafter symbolically indicated as X₂₀.

The peptide may include only the hereinabove noted amino acids or may include additional amino acids at the amino and/or carboxyl end or both the amino and carboxyl end. In general, the peptide does not include more than 40 amino acids.

The peptides including the above basic structure preferably have from 1 to 4 additional amino acids at the amino end.

Accordingly, such preferred peptides may be represented by the structural formula:

    Y.sub.20 --X.sub.20 --

wherein X₂₀ is the hereinabove described basic peptide structure and Y₂₀ is:

(i) R₅ -, or

(ii) R₂ -R₅ -; or

(iii) R₁ -R₂ -R₅ ; or

(iv) R₂ -R₁ -R₂ -R₅ ; preferably Glycine -R₁ -R₂ -R₅.

wherein R₁, R₂ and R₅ are as previously defined.

The carboxyl end of the basic peptide structure may also have additional amino acids which may range from 1 to 13 additional amino acids.

In a preferred embodiment, the basic structure may have from 1 to 7 additional amino acids at the carboxyl end, which may be represented as follows:

-X₂₀ -Z₂₀ wherein

X₂₀ is the hereinabove defined basic peptide structure and Z₂₀ is:

(i) R₁ -, or

(ii) R₁ -R₁ -; or

(iii) R₁ -R₁ -R₄ ; or

(iv) R₁ -R₁ -R₄ -R₄ or

(v) R₁ -R₁ -R₄ -R₄ -R₁ ; or

(vi) R₁ -R₁ -R₄ -R₄ -R₁ -R₆ ; or

(vii) R₁ -R₁ -R₄ -R₄ -R₁ -R₆ -R₆₁ wherein R₁ and R₄ are as previously defined, and R₆ is a neutral hydrophilic amino acid.

Preferred peptides may be represented by the following structural formula

    (Y.sub.20).sub.a --X.sub.20 --(Z.sub.20).sub.b

wherein X₂₀, Y₂₀, and Z₂₀ are as previously defined and a is 0 or 1 and b is 0 or 1.

Representative examples of such peptides comprise the following sequences:

(1) GFASKLGKALKAALKIGANLLGGTPQQ; and

(2) GFGSKLGKALKAALKIGANLLGGTPQQ.

In accordance with yet another aspect of the present invention, there is provided a biologically active amphiphilic peptide selected from the class consisting of:

(1) GFGSFLGKALKAALKIGANMLGGSPQQ;

(2) GFGSFLGKALKAALKIGANMLGGSLQQ;

(3) GFGSFLGLALKAALKIGANMLGGAPQQ;

(4) GLASLLGKALKAALKIGANMLGGSPQQ;

(5) GFGSFLGKALKAALKIGANLLGGTPQQ;

(6) GFAKFLGKALKAALKIGANLLGGTPQQ;

(7) GFGKFLGKALKAALKIGANLLGGTPQQ;

(8) GFKKFLGKALKAALKIGANLLGGTPQQ;

(9) GLASFLGKALKAALKIGANLLGGTPQQ; and

(10) GLASLLGKALKAALKIGANLLGGTPQQ.

Each amino acid residue is a D-amino acid residue or a glycine residue.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula: ##STR8##

Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₂₂ :

R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₁ -R₁.

R₁ is a hydrophobic amino acid, and R₂ is a basic hydrophilic or neutral hydrophilic amino acid.

Each amino acid residue is a D-amino acid residue or a glycine residue.

In one embodiment, the peptide includes the basic structure Y₂₂ -X₂₂ wherein X₂₂ is as hereinabove described and Y₂₂ is:

(i) R₁ ;

(ii) R₂ -R₁ ; or

(iii) R₂ -R₂ -R₁, wherein R₁ and R₂ are as hereinabove described.

The hydrophobic amino may be selected from the class consisting of D-Ala, D-Cys, D-Phe, Gly, D-Ile, D-Leu, D-Met, D-Pro, D-Val, D-Trp and D-Tyr.

The basic hydrophilic amino acid acids may be selected from the class consisting of D-Lys, D-Arg, D-His, D-Orn, D-homoarginine (D-Har), D-2, 4-diaminobutyric acid (D-Dbu), and D-p-aminophenylalanine. The neutral hydrophilic amino acids may be selected from the class consisting of D-Asn, D-Gln, D-Ser, D-Thr, and D-homoserine (D-Hse).

In one embodiment, R₁ is D-leucine. In another embodiment, R₂ is D-lysine. Representative examples of peptides in accordance with this aspect of the present invention include those having the following structures:

(1) LKKLKKLLKLL;

(2) LLKKLKKLLKLL;

(3) KLLKKLKKLLKLL; and

(4) KKLLKKLKKLLKLL.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₂₄ :

R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ wherein R₁ is a hydrophobic amino acid and R₂ is a basic hydrophilic or neutral hydrophilic amino acid. Each amino acid residue is a D-amino acid residue or a glycine residue.

In one embodiment, R₁ is D-leucine. In another embodiment, R₂ is D-lysine.

In one embodiment, the peptide includes the basic structure Y₂₄ -X₂₄, wherein X₂₄ is as hereinabove described, and Y₂₄ is:

(i) R₂ ;

(ii) R₁ -R₂ ;

(iii) R₁ -R₁ -R₂ ;

(iv) R₂ -R₁ -R₁ -R₋₂ ; or

(v) R₂ -R₂ -R₁ -R₁ -R₂.

In one embodiment, the peptide may have the following structure: ##STR9##

In another embodiment, the peptide includes the basic structure X₂₄ -R₂₄, wherein X₂₄ is as hereinabove described, and Z₂₄ is:

(i) R₁ ;

(ii) R₁ -R₁ ;

(iii) R₁ -R₁ -R₂ ;

(iv) R₁ -R₁ -R₂ -R₂ ; or

(v) R₁ -R₁ -R₂ -R₂ -R₁ ;

In one embodiment, the peptide may have the following structure: ##STR10##

In another embodiment, the peptide may include the structure:

(Y₂₄)_(a) - X₂₄ - (Z₂₄)_(b) , wherein X₂₄, Y₂₄ and Z₂₄ are as hereinabove described, and a is 0 or 1, and b is 0 or 1.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₂₆ :

R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₃ wherein R₁ and R₂ are as hereinabove described, and R₃ is a neutral hydrophilic amino acid. Each amino acid residue is a D-amino acid residue or a glycine residue.

In one embodiment, the peptide may have the following structure:

LKKLLKKLKKLLKKN

In one embodiment, the peptide may have the following structure: ##STR11##

In accordance with yet another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₂₈ : R₁ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₄, wherein R₁ and R₂ are as hereinabove described, and R₄ is a neutral hydrophilic amino acid or proline. Each amino acid residue is a D-amino acid residue or a glycine residue.

In one embodiment, the peptide may include the following structure Y₂₈ -X₂₈, wherein X₂₈ is the basic peptide structure hereinabove described, and Y₂₈ is:

(i) -R₁ ;

(ii) -R₁ -R₁ ;

(iii) -R₂ -R₁ -R₁ ;

(iv) -R₁ -R₂ -R₁ -R₁ ;

(v) -R₁ -R₁ -R₂ -R₁ -R₁ ;

(vi) -R₂ -R₁ -R₁ -R₂ -R₁ -R₁ ; or

(vii) -R₂ -R₂ -R₁ -R₁ -R₂ -R₁ -R₁,

wherein R₁ and R₂ are as hereinabove described.

In one embodiment, the peptide may include the structure:

X₂₈ -Z₂₈, wherein X₂₈ is as hereinabove described, and Z₂₈ is:

(i) -R₂ ;

(ii) -R₂ -R₂ ;

(iii) -R₂ -R₂ -R₁ ;

(iv) -R₂ -R₂ -R₁ -R₁ ;

(v) -R₂ -R₂ -R₁ -R₁ -R₂ ;

(vi) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ ; or

(vii) -R₂ -R₂ -R₁ -R₁ -R₂ -R₁ -R₁,

wherein R₁ and R₂ are as hereinabove described.

In one embodiment, the peptide may include the structure:

X₂₈ -Z₂₈, wherein X₂₈ is as hereinabove described, and Z₂₈ is:

(i) -R₂ ;

(ii) -R₂ -R₂ ;

(iii) -R₂ -R₂ -R₁ ;

(iv) -R₂ -R₂ -R₁ -R₁ ;

(v) -R₂ -R₂ -R₁ -R₁ -R₂ ;

(vi) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ ; or

(vii) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁.

In a preferred embodiment, the peptide may have one of the following structures:

(1) LKLLKKLLKKNKKLLKKL; or

(2) LKLLKKLLKKPKKLLKKL.

In another embodiment, the peptide may have the structure (Y₂₈)_(a) -X₂₈ -(Z₂₈)_(b), wherein X₂₈, Y₂₈ and Z₂₈ are as hereinabove described, a is 0 or 1, and b is 0 or 1.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₃₀ :

R₁ -R₁ -R₂ -R₂ R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₃,

wherein R₁, R₂ and R₃ are as hereinabove described. Each amino acid residue is a D-amino acid residue or a glycine residue.

In accordance with another embodiment, the peptide may include the structure Y₃₀ -X₃₀, wherein X₃₀ is as hereinabove described, and Y₃₀ is:

(i) -R₁ ;

(ii) -R₂ -R₁ ;

(iii) -R₂ -R₂ -R₁ ;

(iv) -R₁ -R₂ -R₂ -R₁ ;

(v) -R₁ -R₁ -R₂ -R₂ -R₁ ;

(vi) -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ ; or

(vii) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁, wherein R₁ and R₂ are as hereinabove described.

In another embodiment, the peptide includes the structure X₃₀ -Z₃₀, wherein X₃₀ is as hereinabove described, and Z₃₀ is:

(i) -R₁ ;

(ii) -R₁ -R₅ ;

(iii) -R₁ -R₅ -R₅ ;

(iv) -R₁ -R₅ -R₅ -R₃ ;

(v) -R₁ -R₅ -R₅ -R₃ -R₁ ;

(vi) -R₁ -R₅ -R₅ -R₃ -R₁ -R₃ ;

(vii) -R₁ -R₅ -R₅ -R₃ -R₁ -R₃ -R₃ ;

(viii) -R₁ -R₅ -R₅ -R₃ -R₁ -R₃ -R₃ -R₅ ; or

(ix) -R₁ -R₅ -R₅ -R₃ -R₁ -R₃ -R₃ -R₅ -R₃, wherein R₁ and R₃ are as hereinabove described, and R₅ is proline.

In one embodiment, the peptide has the following structure:

LLKKLKKLLKKLQGPPQGQSPQ.

In one embodiment, the peptide may have the structure (Y₃₀)_(a) -X₃₀ (Z₃₀)_(b), wherein X₃₀, Y₃₀, and Z₃₀ are as hereinabove described, a is 0 or 1, and b is 0 or 1.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following basic structure X₃₂ :

R₁ -R₁ -R₃ -R₂ -R₁ -R₁ -R₁ -R₁ -R₁ -R₁ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -,

wherein R₁, R₂, and R₃ are as hereinabove described. Each amino acid residue is a D-amino acid residue or a glycine residue. In one embodiment, the peptide may have the following structure:

LASKAGAIAGKIAKKLLKKL

In another embodiment, the peptide may include the structure X₃₂ -Z₃₂, wherein X₃₂ is as hereinabove described, and Z₃₂ is:

(i) -R₂ ;

(ii) -R₂ -R₂ ;

(iii) -R₂ -R₂ -R₁ ;

(iv) -R₂ -R₂ -R₁ -R₁ ;

(v) -R₂ -R₂ -R₁ -R₂ ;

(vi) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ ; or

(vii) -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁.

In accordance with yet another aspect of the present invention, there is provided a biologically active amphiphilic peptide having a structure selected from the group consisting of:

(a) R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ ;

(b) R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ ;

(c) R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ ;

(d) R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ ; and

(e) R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁, wherein R₁ and R₂ are as hereinabove described.

In one embodiment, the peptide has the structure (a), and a representative example of such a structure is LKKLKKL.

In another embodiment, the peptide has the structure (b), and a representative example of such a structure is LLKKLKKL.

In another embodiment, the peptide has the structure (c), and a representative example of such a structure is KLLKKLKKL.

In yet another embodiment, the peptide has the structure (d), and a representative example of such a structure is KKLLKKLKKL.

In a further embodiment, the peptide has the structure (e), and representative examples of such a structure are LKKLLKKLKKL and AKKLLKKLKKL as given in the accompanying sequence listing.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula:

LKKLLKKLKKLLKR.

Each amino acid residue is a D-amino acid residue.

In accordance with another aspect of the present invention, there is provided a peptide having the following structural formula: ##STR12## wherein R₁ is Gly or D-Lys; R₂ is D-Ala or D-Lys; R₃ is D-Val or D-Lys; R₄ is D-Ile or D-Glu; and

R₅ is D-Leu or D-Ile. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, the peptide has the following structural formula: ##STR13##

In another embodiment, the peptide has the following structural formula: ##STR14##

In yet another embodiment, the peptide has the following structural formula: ##STR15##

In accordance with another aspect of the present invention, there is provided a peptide having the following structural formula: ##STR16## wherein each of R₁, R₂, R₃, R₄ and R₅ is D-Ile or D-norleucine. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, each of R₁, R₂, R₃ R₄ and R₅ is D-Ile, and such a peptide has the following structural formula: ##STR17##

In another embodiment, each of R₁, R₂, R₃, R₄ and R₅ is D-norleucine, and such a peptide has the following structural formula: ##STR18## wherein Xaa is D-norleucine.

In accordance with another aspect of the present invention, there is provided a peptide having the following structural formula: ##STR19## wherein R is D-Val or D-norleucine. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, R is D-Val, and the peptide has the following structural formula:

GIGKFLKKAKKFGKAFVKIVKK

In another embodiment, R is D-norleucine and the peptide has the following structural formula: ##STR20## wherein Xaa is D-norleucine.

In accordance with yet another aspect of the present invention, there is provided a peptide having the following structural formula: ##STR21## wherein R is D-Arg, D-Asn, or D-homoserine. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, R is D-Arg, and the peptide has the following structural formula:

GIGKFLKKAKKFGKAFVKILKKR

In another embodiment, R is D-Asn, and the peptide has the following structural formula:

GIGKFLKKAKKFGKAFVKILKKN.

In another embodiment, R is D-homoserine, and the peptide has the following structural formula: ##STR22## wherein Xaa is D-homoserine.

In accordance with another aspect of the present invention, there is provided a peptide including the following structural formula: ##STR23## wherein R₁ is D-Met or D-Arg. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, R₁ is D-Met, and the peptide has the following structural formula:

MGIGKFLKKAKKFGKAFVKILKK

In another embodiment, the peptide includes the following structural formula: ##STR24##

In one embodiment, each R₁ is D-Arg, and the peptide has the following structural formula:

RRGIGKFLKKAKKFGKAFVKILKK

In another embodiment, the peptide includes the following structural formula: ##STR25## wherein R₁ is D-Met or D-Arg, and R₂ is a hydrophobic amino acid.

The hydrophobic amino acids are D-Ala, D-Cys, D-Phe, D-Ile, D-Leu, D-Met, Gly, D-Trp, D-Pro, D-Tyr, D-Val, D-norleucine, and D-norvaline.

In one embodiment, R₂ is Gly, and in a preferred embodiment, each R₁ is D-Arg, and the peptide has the following structural formula:

RRGIGKFLKKAKKFGKAFVKILKKG

In accordance with another aspect of the present invention, there is provided a peptide including the following structural formula: ##STR26## wherein R₁ is D-Asp or D-Ile, and R₂ is D-Asp or D-Glu. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, the peptide includes the following structure. ##STR27## wherein R₃ is a hydrophobic amino acid or a basic hydrophilic amino acid.

The basic hydrophilic amino acids are D-Lys, D-Arg, D-His, D-ornithine (D-Orn), D-homoarginine (D-Har), D-2,4-diaminobutyric acid (D-Dbu), and D-p-aminophenylalanine.

In one embodiment, the peptide has the following structural formula:

GIGKFLKKAKKFGKAFVKILKKDDK

In another embodiment, the peptide includes the following structure: ##STR28## wherein R₄ is a basic hydrophilic amino acid.

In one embodiment, the peptide has the following structural formula:

GIGKFLKKAKKFGKAFVKILKKIEGR

In accordance with another aspect of the present invention, there is provided a peptide selected from the group consisting of:

(1) GIIGKFLKKAKKFGKAFVKILKK;

(2) GIGKFLKLAKKFGKAFVKILKK;

(3) GIGKFLKKAKKGIGAVLKVLTTGK;

(4) GIGKFLKKAKKFGKAFIKILKK;

(5) GIGKFLKKAKKFG(Har)AFVKILKK;

(6) GIGKFLKKAKKFGKAFVKILKK;

(7) GGKFLKKAKKFGKAFVKILKK; and

(8) FGKILKKAKKIGKAIVKFLKK.

Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula X₃₄ :

R₁ -R₁ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂,

wherein R₁ is a hydrophobic amino acid, and R₂ is a basic hydrophilic or neutral hydrophilic amino acid. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

The hydrophobic amino acids are selected from the group consisting of D-Ala, D-Cys, D-Phe, Gly, D-Ile, D-Leu, D-Met, D-methionine sulfoxide, D-Val, D-Trp, and D-Tyr.

The basic hydrophilic amino acids are selected from the group consisting of D-Lys, D-Arg, D-His, D-Orn, D-homoarginine (D-Har), D-2,4-diaminobutyric acid (Dbu), and D-p-aminophenylalanine.

The neutral hydrophilic amino acids are selected from the group consisting of D-Asn, D-Gln, D-Ser, and D-Thr.

In accordance with one embodiment, R₁ is D-leucine. In accordance with another embodiment, R₂ is D-lysine.

In a preferred embodiment, the peptide has the following structure (I):

I. ##STR29##

In accordance with another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula X₃₆ :

R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁,

wherein R₁ and R₂ are as hereinabove described. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In a preferred embodiment, the peptide has the following structure (II):

II. ##STR30##

In accordance with yet another aspect of the present invention, there is provided a biologically active amphiphilic peptide having the following structural formula X₃₈ :

R₁ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁, wherein R₁ is a hydrophobic amino acid, and R₂ is a basic hydrophilic or neutral hydrophilic amino acid as before defined. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

Preferably, the peptide has the following structure (III): ##STR31##

In accordance with another aspect of the present invention, there is provided a compound comprising an analogue of Peptide III, said peptide being in an amide-or carboxy-terminated (preferably amide-terminated) form. The Peptide III, also hereinafter sometimes referred to as the "parent peptide," is represented by the following structural formula, and the numbers below each amino acid residue refer to the position of the residue in the peptide. ##STR32##

The parent peptide is substituted in at least one of positions 1, 3, 4 and 7-18 as follows:

    ______________________________________                                         Residue No.                                                                               Substituent                                                         ______________________________________                                         1          D-Methionine sulfoxide, D-Lys, or D-Met                             3          D-Methionine sulfoxide, D-Lys, or D-Met                             4          D-Methionine sulfoxide, D-Lys, D-Met, D-His,                                   D-Ser, or D-Arg                                                     7          D-Methionine sulfoxide, D-Lys, or D-Met                             8          D-Methionine sulfoxide, D-Lys, or D-Met                             9          D-Methionine sulfoxide                                              10         D-Methionine sulfoxide                                              11         D-Methionine sulfoxide, D-Met, D-Ser, D-Lys,                                   D-Arg, D-His or Gly                                                 12         D-Methionine sulfoxide                                              13         D-Methionine sulfoxide, or D-Met                                    14         D-Methionine sulfoxide, D-Lys, or D-Met                             15         D-Methionine sulfoxide, D-Lys, or D-Met                             16         D-Methionine sulfoxide                                              17         D-Methionine sulfoxide                                              18         D-Methionine sulfoxide, or D-Met                                    ______________________________________                                    

Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In accordance with one embodiment, at least one of amino acid residues, 1, 7, 8, 11, 14, 15, and 18 can be substituted with D-methionine sulfoxide.

In accordance with another embodiment, at least one of amino acid residues 1, 7, 8, 14, 15, and 18 can be substituted with a D-methionine residue.

In accordance with yet another embodiment, at least one of amino acid residues 4, 7, 8, 11, and 14 can be substituted with a D-lysine residue.

In accordance with a further embodiment, amino acid residue 4 is substituted with a D-lysine residue, and amino acid residue 11 is substituted with a D-methionine residue.

In accordance with another embodiment, at least one of amino acid residues 4 and 11 can be substituted with a D-arginine residue.

In accordance with yet another embodiment, at least one of amino acid residues 4 and 11 can be substituted with a D-histidine residue.

In accordance with another embodiment, amino acid residue 11 is substituted with a glycine residue.

In accordance with another aspect of the present invention, there is provided a compound comprising an analogue of the parent peptide hereinabove described, said peptide being in an amide- or carboxy-terminated (preferably amide-terminated) form, wherein at least one of the amino acid residues 1 through 7, 9, 11, 12, 14, 16 or 18 is deleted from the parent peptide. Each amino acid residue is a D-amino acid residue. In one embodiment, at least one of amino acid residues 3, 7, 11, 14, or 18 is deleted from the parent peptide. In other embodiments, amino acid residues 1 through 3, 1 through 4, 1 through 5, 1 through 6, and 1 through 7 are deleted from the peptide.

Representative examples of such peptides include the following: ##STR33##

In accordance with a further aspect of the present invention, there is provided a biologically active amphiphilic peptide which includes the following structural formula X₄₀ :

R₁ -R₁ -R₂ -R₂ -R₁ -R₂ -R₂ -R₁ -R₁ -R₂ -R₂ -R₁ -R₁,

wherein R₁ and R₂ are as hereinabove described. Each amino acid residue which is not a glycine residue is a D-amino acid residue.

In one embodiment, the peptide can include the following structure:

X₄₀ -Z₄₀, wherein X₄₀ is as hereinabove described, and Z₄₀ is:

(i) R₂ ;

(ii) R₂ -R₁ ; or

(iii) R₂ -R₁ -R₁.

In one embodiment, the peptide has the following structural formula: ##STR34##

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 demonstrates that administration of a D-amino acid peptide delayed and reduced the accumulation of ascites in mice compared to controls after injection of Spontaneous Ovarian Teratoma cells.

Applicants unexpectedly have found that the above-mentioned peptides, all of which consist entirely of D-amino acids or glycine residues, have biological activity.

The above-mentioned peptides also have increased resistance to proteolytic enzymes while retaining their biological activity. Therefore, such peptides may be administered orally.

In general the peptides hereinabove described are ion channel-forming peptides. Thus, in accordance with an aspect of the present invention, there is provided a biologically active amphiphilic peptide or protein. The peptide or protein is an ion channel-forming peptide or protein. At least one amino acid residue of the peptide or protein is optically active, and each optically active amino acid residue is a D-amino acid residue.

The term "optically active" as used herein means that the amino acid residue may exist in the D-form or the L-form. It is to be noted, however, that glycine is not optically active; however, glycine may be included in the peptide or protein if all other amino acid residues in the peptide or protein are D-amino acid residues.

An ion channel-forming peptide or protein or ionophore is a peptide or protein which increases the permeability of ions across a natural or synthetic lipid membrane. B. Christensen, et al., PNAS, Vol. 85, pgs. 5072-5076 (July 1988) describes methodology which indicates whether or not a peptide has ion channel-forming properties and is therefore an ionophore. As used herein, an ion channel-forming peptide or ion channel-forming protein is a peptide or protein which has ion channel-forming properties as determined by the method of Christensen, et al.

An amphiphilic peptide or protein is a peptide or protein which includes both hydrophobic and hydrophilic regions.

In general, the peptides hereinabove described, and/or analogues or derivatives thereof are generally water soluble to a concentration of at least 20 mg/ml at neutral pH in water. In addition, such peptides are non-hemolytic; i.e., they will not rupture blood cells at effective concentrations. The peptides cause less than 5% hemolysis of human erythrocytes at a concentration of 500 μg/ml. In addition, the structure of such peptide provides for flexibility of the peptide molecule. When the peptide is placed in water, it does not assume an amphiphilic structure. When the peptide encounters an oily surface or membrane, the peptide chain folds upon itself into a rod-like structure.

The peptides may be C-terminal acids or amides.

The peptides may be administered to a host; for example a human or non-human animal, in an amount effective to inhibit growth of a target cell or virus. Thus, for example, the peptides and/or analogues or derivatives thereof may be used as antimicrobial agents, anti-viral agents, antibiotics, anti-tumor agents, antiparasitic agents, spermicides, as well as exhibiting other bioactive functions.

The term "antimicrobial" as used herein means that the peptides of the present invention inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, or the like.

The term "antibiotic" as used herein means that the peptides employed in the present invention produce effects adverse to the normal biological functions of the cell, tissue, or organism including death or destruction and prevention of the growth or proliferation of the biological system when contacted with the peptides.

The term "spermicidal" as used herein means that the peptides employed in the present invention, inhibit, prevent, or destroy the motility of sperm.

The term "antiviral" as used herein means that the peptides employed in the present invention inhibit, prevent, or destroy the growth or proliferation of viruses.

The term "anti-tumor" as used herein means that the peptide inhibits the growth of or destroys tumors.

The term "antiparasitic" as used herein means that the peptides of the present invention may be used to inhibit the growth of or destroy parasites.

The peptides of the present invention have a broad range of potent antibiotic activity against a plurality of microorganisms including Gram-positive and Gram-negative bacteria, fungi, protozoa, and the like, as well as parasites. The peptides of the present invention allow a method for treating or controlling microbial infection caused by organisms which are sensitive to the peptides. Such treatment may comprise administering to a host organism or tissue susceptible to or affiliated with a microbial infection an antimicrobial amount of at least one of the peptides.

Because of the antibiotic properties of the peptides, they may also be used as preservatives or sterilants of materials susceptible to microbial contamination.

The peptide may be administered in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution. Such pharmaceutical compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule, or the like. The peptide compositions may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, and the like, as well as by parasites.

The peptides of the present invention may be administered to a host; in particular an animal, in an effective antibiotic and/or anti-tumor and/or anti-viral and/or anti-microbial and/or anti-parasitic and/or an antispermicidal amount.

Depending on the use, a composition in accordance with the invention will contain an effective anti-microbial amount and/or an effective antispermicidal amount and/or an effective anti-viral amount and/or an effective anti-tumor amount and/or an effective antibiotic amount and/or anti-parasitic amount of one or more of the hereinabove described peptides which have such activity.

The peptide of the present invention may also be employed in promoting or stimulating healing of a wound in a host.

The term "wound healing" as used herein includes various aspects of the would healing process.

These aspects include, but are not limited to, increased contraction of the wound, increased deposition of connective tissue, as evidenced by, for example, increased deposition of collagen in the wound, and increased tensile strength of the wound, i.e., the peptides increase wound breaking strength. The peptides of the present invention may also be employed so as to prevent the inhibition of wound healing in individuals having a compromised immune system.

The peptides of the present invention may be used in the treatment of external burns and to treat and/or prevent skin and burn infections. In particular, the peptides may be used to treat skin and burn infections caused by organisms such as, but i not limited to, P. aeruginosa and S. aureus.

The peptides are also useful in the prevention or treatment of eye infections. Such infections may be caused by bacteria such as, but not limited to, P. aerurinosa, S. aureus and N. gonorrhoeae, by fungi such as but not limited to C. albicans and A. fumigatus, by parasites such as but not limited to A. castellani, or by viruses.

The peptides may also be effective in killing cysts, spores, or trophozoites of infection-causing organisms. Such organisms include, but are not limited to Acanthamoeba which forms trophozoites or cysts, C. albicans, which forms spores, and A. fumigatus, which forms spores as well.

In general, the peptide is employed to provide peptide dosages of from 0.1 mg. to 500 mg. per kilogram of host weight. When administered topically, the peptide is used in a concentration of from 0.05% to 10%.

The peptides may be produced by known techniques and obtained in substantially pure form. For example, the peptides may be synthesized on an automatic synthesizer. Journal of the American Chemical Society, Vol. 85, pgs. 2149-54 (1963). It is also possible to produce such peptides by genetic engineering techniques.

The peptides of the present invention may be administered alone, or in combination with other such peptides. The peptides may also be administered in combination with biologically active peptides or proteins containing L-amino acid residues, such as ion channel-forming proteins, or the peptides of the present invention may be administered in combination with other active components, such as ions having pharmacological properties or antibiotics.

Biologically active peptides containing L-amino acid residues which may be administered in combination with the peptides of the present invention include magainin peptides, PGLa peptides, XPF peptides, cecropins, and sarcotoxins. Magainin peptides are described in Proc. Natl. Acad. Sci., Vol. 84, pgs 5449-53 (August 1987). A review of XPF and PGLa peptides may be found in Hoffman, et al., EMBOJ., 2:711-714 (1983). Andreu, et al., J. Biochem, 149:531-535 (1985); Gibson, et al., Biochem J., 243:113-120 (1987).

The cecropins and analogues and derivatives thereof are described in Ann. Rev. Microbiol., Vol. 41, pgs, 103-26 (1987), and Christensen, et al., PNAS, Vol. 85, pgs. 5072-5076. (1988). Sarcotoxins and analogues and derivatives thereof are described in Molecular Entomotogy, pgs 369-78, Alan R. Liss, Inc. (1987).

Ion channel-forming proteins or peptides which may be employed in combination with the peptides of the present invention include defensins, also known as human neutrophil antimicrobial peptides (HNP), major basic protein (MBP) of eosinophils, bactericidal permeabil.ity-increasing protein (BPI), and a pore-forming cytotoxin called variously perforin, cytolysin, or pore-forming protein. Defensins are described in Selsted, et al., J. Clin. Invest., Vol. 76, pgs. 1436-1439 (1985). MBP proteins are described in Wasmoen, et al., J. Biol. Chem., Vol. 263, pgs. 12559-12563 (1988). BPI proteins are described in Ooi, et al., J. Biol. Chem. Vol. 262, pgs. 14891-14894 (1987). Perforin is described in Henkart, et al., J. Exp. Med., 160:75 (1984), and in Podack, et al., J. Exp. Med., 160:695 (1984). The above articles are hereby incorporated by reference.

Ions having pharmacological properties which may be employed in combination with the peptides of the present invention include anions such as fluoride, peroxide, and bicarbonate anions, and cations such as silver, zinc, mercury, arsenic, copper, platinum, antimony, gold, thallium, nickel, selenium, bismuth, and cadmium cations.

A ion having pharmacological properties is one which when introduced into a target cell inhibits and/or prevents and/or destroys the growth of the target cell.

Such an ion having pharmacological properties is one which in the absence of an ion channel forming peptide is unable to cross a natural or synthetic lipid membrane; in particular a cell membrane, in sufficient amounts to affect a cell adversely.

The peptide and the ion having pharmacological properties, whether administered or prepared in a single composition or in separate compositions, are employed in amounts effective to inhibit and/or prevent and/or destroy the growth of the target cell. In effect, the ion potentiates the action of the peptide, i.e., the amount of ion is effective to reduce the maximum effective concentration of the peptide or protein for inhibiting growth of a target cell.

Antibiotics which may be employed in combination with the peptides of the present invention include bacitracins, aminoglycosides, hydrophobic antibiotics, including macrolide antibiotics, penicillins, monobactams, or derivatives or analogues thereof.

Other antibiotics which may be used (whether or not hydrophobic) in combination with the peptides of the present invention are antibiotics which are 50-S ribosome inhibitors such as lincomycin; clindamycin; and chloramphenicol; etc.; and antibiotics which have a large lipid like lactone ring, such as mystatin; pimaricin, etc.

The peptide and antibiotic may be administered by direct administration to a target cell or by systemic or topical administration to a host which includes the target cell, in order to prevent, destroy or inhibit the growth of a target cell. Target cells whose growth may be prevented, inhibited, or destroyed by the administration of the peptides and antibiotic include Gram-positive and Gram-negative bacteria as well as fungal cells.

The antibiotic, such as those hereinabove described, or derivatives or analogues thereof, when used topically, is generally employed in a concentration of about 0.1% to about 10%. When used systemically, the antibiotic or derivative or analogue thereof is generally employed in an amount of from 1.25 mg. to about 45 mg. per kg. of host weight per day. Peptide dosages may be those as hereinabove described.

As representative examples of administering the peptide and antibiotic for topical or local administration, the peptide could be administered in an amount of from about 0.1% to about 10% weight to weight, and the antibiotic is delivered in an amount of from about 0.1% to about 10% weight to weight.

In accordance with another embodiment, the peptides of the present invention may be administered in combination with an antiparasitic agent or an antifungal agent.

Antiparasitic agents which may be employed include, but are not limited to, anti-protozoan agents. Examples of specific anti-parasitic agents which may be employed include, but are not limited to, pentamidine isethionate, and propamidine isethionate (Brolene).

Anti-fungal agents which may be employed include, but are not limited to, ketoconazole. It is also to be understood that certain anti-parasitic agents may also have anti-fungal activity, and that certain anti-fungal agents may have anti-parasitic activity.

In accordance with another embodiment, the peptides of the present invention may be administered in combination with an antibiotic which inhibits DNA gyrase, which is an enzyme involved in the formation of bonds between individual coiling strands of replicating bacterial DNA. Thus, DNA gyrase is necessary for the normal replication of bacterial DNA, and, therefore, antibiotics which inhibit DNA gyrase inhibit the normal replication of bacterial DNA.

Examples of antibiotics which inhibit DNA gyrase include nalidixic acid, oxolinic acid, cinoxacin, and quinolone antibiotics which include ciprofloxacin, norfloxacin, ofloxacin, enoxacin, pefloxacin, lomefloxacin, fleroxacin, tosulfloxacin, temafloxacin, and rufloxacin.

In accordance with another embodiment, the peptides of the present invention may be administered for the purpose hereinabove described in combination with other biologically active amphiphilic peptides, or in combination with ion channel-forming proteins.

The invention will now be further described with respect to the following examples; however, the scope of the present invention is not to be limited thereby.

In the following examples, the term "D" indicates that each amino acid residue is a D-amino acid residue or a glycine residue, and the term "Oct" indicates that the peptide is substituted at the N-terminal with an octanoyl group.

EXAMPLE 1

The procedure for the following antibacterial assay is based upon the guidelines of the National Committee for Clinical Laboratory Standards, Document M7-T2, Volume 8, No. 8, 1988.

Stock solutions of the following peptides:

(SEQ ID NO:1)-NH₂

D-(SEQ ID NO:1)-NH₂

(SEQ ID NO:2)-NH₂

D-(SEQ ID NO:2)-NH₂

(SEQ ID NO:3)-NH₂

D-(SEQ ID NO:3)-NH₂

(SEQ ID NO:4)-NH₂

D-(SEQ ID NO:4)-NH₂

(SEQ ID NO:5)-NH₂

D-(SEQ ID NO:5)-NH₂

(SEQ ID NO:6)-NH₂

D-(SEQ ID NO:6)-NH₂

(SEQ ID NO:7)-NH₂

D-(SEQ ID NO:7)-NH₂

(SEQ ID NO:8)-NH₂

D-(SEQ ID NO:8)-NH₂

(SEQ ID NO:9)-NH₂

D-(SEQ ID NO:9)-NH₂

(SEQ ID NO:10)-NH₂

D-(SEQ ID NO:10)-NH₂

Oct-(SEQ ID NO:1l)-NH₂

Oct-D-(SEQ ID NO:l1)-NH₂

were prepared at a concentration of 512 μpg/ml in sterile deionized distilled water and stored at -70° C. The "D" indicates that each amino acid residue of the peptide is a D-amino acid residue or a glycine residue.

The stock peptide solutions are diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg/ml. 1.5×10⁵ CFUs/ml of either S. aureus ATCC 25923, E. coli ATCC 25922, P. aeruginosa ATCC 27853, or C. albicans were added to the wells in full strength Mueller Hinton broth (BBL 11443) from a mid-log culture. The inoculums are standarized spectrophotometrically at 600 nm and is verified by colony counts. The plates are incubated for 16-20hours at 37° C., and the minimal inhibitory concentration (MIC) for each peptide is determined. Minimal inhibitory concentration is defined as the lowest concentration of peptide which produces a clear well in the microtiter plate. The MIC values for each peptide are given in Table I below:

                  TABLE I                                                          ______________________________________                                         MIC (μg/ml)                                                                                S.      P.        E.    C.                                      Peptide        aureus  aeruginosa                                                                               coli  albicans                                ______________________________________                                         (SEQ ID NO:1)-NH.sub.2                                                                        8       16        4     N/A                                     D-(SEQ ID NO:1)-NH.sub.2                                                                      8       32        4     N/A                                     (SEQ ID NO:2)-NH.sub.2                                                                        4       2         4     64                                      D-(SEQ ID NO:2)-NH.sub.2                                                                      4       4         4     32                                      (SEQ ID NO:3)-NH.sub.2                                                                        8       8         4     32                                      D-(SEQ ID NO:3)-NH.sub.2                                                                      8       4         2     N/A                                     (SEQ ID NO:4)-NH.sub.2                                                                        4       4         4     N/A                                     D-(SEQ ID NO:4)-NH.sub.2                                                                      8       8         4     32                                      (SEQ ID NO:5)-NH.sub.2                                                                        16      32        4     N/A                                     D-(SEQ ID NO:5)-NH.sub.2                                                                      32      32        4     32                                      (SEQ ID NO:6)-NH.sub.2                                                                        4       8         2     32                                      D-(SEQ ID NO:6)-NH.sub.2                                                                      4       8         2     16                                      (SEQ ID NO:7)-NH.sub.2                                                                        4       4         2     64                                      D-(SEQ ID NO:7)-NH.sub.2                                                                      4       4         2     16                                      (SEQ ID NO:8)-NH.sub.2                                                                        32      >256      256   256                                     D-(SEQ ID NO:8)-NH.sub.2                                                                      16      >256      >256  256                                     (SEQ ID NO:9)-NH.sub.2                                                                        16      8         32    32                                      D-(SEQ ID NO:9)-NH.sub.2                                                                      32      8         32    32                                      (SEQ ID NO:10)-NH.sub.2                                                                       16      8         8     32                                      D-(SEQ ID NO:10)-NH.sub.2                                                                     8       8         16    32                                      Oct-(SEQ ID NO:11)-NH.sub.2                                                                   4       4         8     32                                      Oct-D-(SEQ ID NO:11)-NH.sub.2                                                                 4       4         8     32                                      ______________________________________                                    

The above results indicate that when all residues of the biologically active peptide are D-amino acid residues or glycine residues, the peptides retain their biological activity.

EXAMPLE 2

50 μl of prereduced brain-heart infusion broth (BHI broth) is added to each well of a 96-well microtiter plate. Stock solutions of (SEQ ID NO:8)-NH₂ and D-(SEQ ID NO:8)-NH₂ are diluted in serial dilutions (1:2) down the wells of a microtiter plate so that the final concentrations of the peptides in the wells are 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, and 256 μg/ml. Prereduced saline, vancomycin, and metronidazole are used as controls in the same concentrations. Following the serial dilutions of the antimicrobial agents and the peptides, 100μl of prereduced BHI broth is added to every well and the plate is incubated overnight in an anaerobic chamber. Inoculums of C. difficile, B. fragilis, L. fermentum, and E. coli were obtained from overnight cultures in BHI under anaerobic conditions. A 1:100 dilution of the overnight cultures were made in prereduced saline; 0.5 ml of the 1:100 dilution of each of the overnight cultures then was added to 9.5 ml saline to create concentrations of approximately 10⁵ organisms per ml. 50 μl of the cell suspensions then were added to each well. One row of wells serves as a control, and receives no organisms; another row of wells receives only cells without antimicrobial agents or peptides. The plates are incubated anaerobically for 24 to 36 hours, and then visual endpoints are read either as "positive " or "negative." The minimum inhibitory concentration (MIC) is the concentration where no turbidity is observed. The MIC values for each peptide against each organism are given in Table II below.

                  TABLE II                                                         ______________________________________                                                      MIC (μg/ml)                                                                 C.       B        L.      E.                                      Peptide      difficile                                                                               fragilis fermentum                                                                              coli                                    ______________________________________                                         (SEQ ID NO:8)-NH.sub.2                                                                      1        2        8       256                                     D-(SEQ ID NO:8)-NH.sub.2                                                                    1        1        1       >256                                    ______________________________________                                    

EXAMPLE 3

Stock cultures of P.gingivalis strains 381; A7AI-28; FAY-19M-1; and W-50 are maintained on Brucella blood agar plates with hemin and vitamin K₁ (BBL, Cockeysville, Md.), grown under anaerobic conditions (Coy Anaerobic Chambers, Ann Arbor, Mich.) with an atmosphere of 80% N₂, 10% H₂, and 10% CO₂ at 37° C. The cultures then are grown up in brain heart infusion broth (BHI broth) (BBL, Cockeysville, Md.), plus hemin (2.5 mg/1) (Sigma Chemical Co., St. Louis, Mo.) plus 0.25 mg/l vitamin K₁ (Sigma Chemical Co.). For antimicrobial susceptibility testing, cultures are taken from overnight (24 hour) broth cultures and diluted in fresh BHI broth (plus hemin and vitamin K₁) to deliver 1×10⁶ colony forming units (CFU's) per ml in each microtiter test well.

Antimicrobial susceptibility testing is performed according to the guidelines of the National Committee for Clinical Laboratory Standards, Document M11-T2 (1989). Microtiter plates (Corning, Corning, N.Y.) are aseptically filled with BHI broth (plus hemin and vitamin K₁) to a volume of 100μl with a Beckman Biomek 1000 robotic instrument (Beckman Instruments, Palo Alto, Calif.). Peptides (SEQ ID NO:1)-NH₂, D-(SEQ ID NO:1)-NH₂, (SEQ ID NO:6)-NH₂, D-(SEQ ID NO:6)-NH₂, (SEQ ID NO:7)-NH₂, and D-(SEQ ID NO:7)-NH₂, are tested in duplicate lanes by adding manually 100μl of a 1.024 mg/ml peptide solution in water (wt./vol.) to the top wells of a microtiter plate. The peptide is serially diluted 1:2 by mixing and transferring 100 μl from the top well down to the bottom well in the lane with the Beckman Biomek 1000. The last 100 μl from the bottom well is discarded. 100μl of bacteria is added in BHI broth (plus hemin and vitamin K₁) to each test well to give final peptide concentrations of 256 μg/ml, 128 μg/ml, 64 μg/ml, 32 μg/ml, 16 μg/ml, 8 μg/ml, 4 μg/ml, 2 μg/ml, 1 μg/ml, 0.50 μg/ml, and 0.25 μg/ml. The plates are incubated in an anaerobic chamber at 37° C. for 24 to 48 hours. After incubation, the minimum inhibitory concentration (MIC) is determined as the lowest concentration of peptide which inhibits growth as determined by visual inspection and optical density when read on a Dynatech MR5000 (Dynatech Laboratories, Chantilly, Va.) microtiter plate reader at 630 nm. The MIC values for each peptide against the various P. gingivalis strains are given in Table III below.

                  TABLE III                                                        ______________________________________                                                        MIC (μg/ml)                                                                 P. gingivalis strain                                            Peptide       381    A7AI-28   FAY-19M-1                                                                              W-50                                    ______________________________________                                         (SEQ ID NO:1)-NH.sub.2                                                                       256    64        64      32                                      D-(SEQ ID NO:1)-NH.sub.2                                                                     256    32        64      8                                       (SEQ ID NO:6)-NH.sub.2                                                                       64     64        128     256                                     D-(SEQ ID NO:6)-NH.sub.2                                                                     2      8         8       8                                       (SEQ ID NO:7)-NH.sub.2                                                                       256    2         32      8                                       D-(SEQ ID NO:7)-NH.sub.2                                                                     32     2         8       2                                       ______________________________________                                    

EXAMPLE 4

Melanoma cell lines WM-239-A (metastatic melanoma), WM278 (primary melanoma), WM793 (primary melanoma) and WM1158 (metastatic melanoma) were seeded in 24-well plates at a concentration of 7.5×10⁴ cells/ml containing 2% tumor medium.

Peptides (SEQ ID NO:6) and D-(SEQ ID NO:6), in which each amino acid residue is a D-amino residue or a glycine residue, were frozen at -20° C. in 100 ml aliquots at a concentration of 25 mg/ml. Each peptide was then dissolved in sterile distilled water at a dilution of 1:1,000 or 1:5,000 to provide peptide concentrations of 25 μg/ml or 5 μg/ml, respectively. The peptides at the 25 μg/ml or 5 μg/ml concentrations were then added to the plates containing the melanoma cells. The amount of ³ H-thymidine incorporation for each well was taken on days 1, 3, and 7. The percentage of growth reduction of each cell line after 7 days by each peptide is given in Table IV below.

                  TABLE IV                                                         ______________________________________                                         Dose        % Growth Reduction (7 days)                                        Peptide (μg/ml)                                                                             WM239-A   WM-278 WM793  WM1158                                 ______________________________________                                         (SEQ ID 5       0         0      11.6   0                                      NO:6)   25      95.8      96.0   98.0   98.5                                   D-(SEQ ID                                                                              5       82.0      93.1   95.6   98.0                                   NO:6)   25      96.3      96.1   98.4   98.8                                   ______________________________________                                    

EXAMPLE 5

Peptides (SEQ ID NO:10) and D-(SEQ ID NO:10), in which each amino acid is a D-amino acid residue, are tested for % growth reduction of melanoma cell lines WM852 (grown in 2% tumor media or PF media), WM983A (grown in 2% tumor media or PF media), WM793 (grown in 2% tumor media), and WM1985 (grown in PF media) according to the procedure of Example 4, except that only a concentration of 25 μg/ml was employed for (SEQ ID NO:10), and concentrations of 1.0, 2.5, 5.0, 10.0, and 25.0 μg/ml were employed for D-(SEQ ID NO:10). The results are given in Table V below.

                                      TABLE V                                      __________________________________________________________________________     Dose      % Growth Reduction (7 days)                                          Peptide                                                                              (μg/ml)                                                                         WM852-2%                                                                             WM-983A-2%                                                                            WM793-2%                                                                             WM852-PF                                                                             WM983A-PF                                                                             WM1985-PF                            __________________________________________________________________________     (SEQ ID                                                                              25  0     0      8.3   54.0  60.5   43.0                                 NO:10)                                                                         D-(SEQ ID                                                                            1.0 3.0   0      N/A   2.9   3.3    5.5                                  NO:10)                                                                               2.5 5.4   0      18.2  22.9  13.9   23.6                                       5.0 8.8   0      0     32.4  29.8   52.1                                       10.0                                                                               16.2  13.8   12.3  80.6  84.9   84.3                                       25.0                                                                               84.7  71.3   84.9  91.1  90.0   92.6                                 __________________________________________________________________________

EXAMPLE 6

The following melanoma cell lines are seeded in 200 ul of 2.0% tumor medium at a density of 1.5×10⁴ cells per well in a 96-well microtiter plate:

WM1791A

WM35

WM852

WM983A

WM1366

WM8

WM115

WM983B

WM39

WM1158

SKMel 23

WM164

WM902B

WM793; and

WM239A.

The following day, the media are removed, and solutions of peptides D-(SEQ ID NO:6) and D-(SEQ ID NO:10) in 2% tumor media are added in triplicate at the following concentrations:

0 (control)

1.0 μg/ml

5.0 μg/ml

25.0 μg/ml

On the next day, 1 μCi of 3H-thymidine is added to each well. After 18 hours, the medium was discarded, and the cells were detached by addition of 50 ul of trypsin/versene solution. The loose cells were then harvested using a Tomtec cell harvester. The cells were trapped on filters, and the radioactivity quantitated by liquid scintillation counting using a Packard instrument. The radioactive counts for each cell line contacted with the peptides is given in Table VI below.

                                      TABLE VI                                     __________________________________________________________________________     Dose     Radioactive Counts                                                    Peptide                                                                              μg/ml                                                                          WM1791A                                                                              WM35                                                                               WM852                                                                               WM983A                                                                              WM1366                                                                              WM115                                                                               WM983B                                  __________________________________________________________________________     D-(SEQ ID                                                                            0  8068  74761                                                                              198000                                                                              389000                                                                              307000                                                                              296000                                                                              202000                                  NO:6) 1  5655  47241                                                                              169000                                                                              335000                                                                              288000                                                                              222000                                                                              225000                                        5  1065  3187                                                                               2699 5598 1910 373  135                                           25 456   360 554  313  1692 263  115                                     D-(SEQ ID                                                                            0  9359  76294                                                                              191000                                                                              300000                                                                              303000                                                                              298000                                                                              211000                                  NO:10)                                                                               1  6188  44803                                                                              163000                                                                              217000                                                                              366000                                                                              213000                                                                              195000                                        5  3318  8779                                                                               109180                                                                              49809                                                                               327000                                                                              37989                                                                               45654                                         25 642   333 1083 304  787  424  104                                     __________________________________________________________________________     Dose     Radioactive Counts                                                    Peptide                                                                              μg/ml                                                                          WM39  WM8 WM1158                                                                              SkMe123                                                                             WM164                                                                               WM902B                                                                              WM973                                                                               WM239A                             __________________________________________________________________________     D-(SEQ ID                                                                            0  27768 17189                                                                              159000                                                                              142000                                                                              153000                                                                              54570                                                                               88754                                                                               37986                              NO:6) 1  27635 19226                                                                              131000                                                                              114846                                                                              158000                                                                              49299                                                                               81675                                                                               38053                                    5  1308  248 884  2028 4143 150  7284 221                                      25 273   99  2108 429  331  75   770  459                                D-(SEQ ID                                                                            0  27887 19099                                                                              144000                                                                              141000                                                                              142000                                                                              55940                                                                               88816                                                                               42505                              NO:10)                                                                               1  31033 21340                                                                              130000                                                                              114000                                                                              162000                                                                              47809                                                                               94365                                                                               33359                                    5  13760 3320                                                                               135000                                                                              64253                                                                               17235                                                                               23390                                                                               87751                                                                               8649                                     25 199   13  4949 377  182  72   257  15                                 __________________________________________________________________________

EXAMPLE 7

Groups of mice, with 5 or 6 mice in each group, were injected with Spontaneous Ovarian Teratoma Cells. At 2 days and 5 days after the injection of the Spontaneous Ovarian Teratoma Cells (SOT cells), the groups of mice were injected intraperitoneally with a control saline solution, adriamycin, or with one of the following peptides:

(SEQ ID NO:6)-NH₂

D-(SEQ ID NO:6)-NH₂

(SEQ ID NO:7)-NH₂

D-(SEQ ID NO:7)-NH₂

The mice in each group were then monitored for median survival time (in days) and for increase in life span (ILS) vis-a-vis the control group. The median survival time is the day of death of the third mouse in each group. The result for the groups of mice given intraperitoneal injections of peptide at days 2 and 5 following injection of SOT cells are given in Table VII below.

                  TABLE VII                                                        ______________________________________                                                      Dose       Median Survival                                        Peptide      (mg/kg/day)                                                                               Time (days) ILS                                        ______________________________________                                         (SEQ ID NO:6)-NH.sub.2                                                                      60/50      >42(21)     100%                                       D-(SEQ ID NO:6)-NH.sub.2                                                                    45         38(21)      83%                                        D-(SEQ ID NO:6)-NH.sub.2                                                                    15         35(21)      67%                                        (SEQ ID NO:7)-NH.sub.2                                                                      22         38(21)      81%                                        D-(SEQ ID NO:7)-NH.sub.2                                                                    22         41.5(17)    144%                                       ______________________________________                                    

In other experiments the mice are not treated with peptide (SEQ ID NO:7)-NH₂ or D-(SEQ ID NO:7)-NH₂ in an amount of 10 mg/kg until day five after injection of the Spontaneous Ovarian Teratoma Cells. As shown in FIG. 1, the body weight data indicates that the accumulation of ascites fluid in mice treated with D-(SEQ ID NO:7)-NH₂ was delayed and was also significantly less than the control group which was given a saline solution or for animals treated with (SEQ ID NO:7)-NH₂.

The peptides of the present invention, whether administered alone or in combination with agents such as toxic ions, antibiotics, or other biologically active peptides or proteins as hereinabove described, may be employed in a wide variety of pharmaceutical compositions in combination with a non-toxic pharmaceutical carrier or vehicle such as a filler, non-toxic buffer, or physiological saline solution. Such pharmaceutical compositions may be used topically or systemically and may be in any suitable form such as a liquid, solid, semi-solid, injectable solution, tablet, ointment, lotion, paste, capsule or the like. The peptide and/or agent as hereinabove described may also be used in combination with adjuvants, protease inhibitors, or compatible drugs where such a combination is seen to be desirable or advantageous in controlling infection caused by harmful microorganisms including protozoa, viruses, and the like.

The peptide may be administered to a host, in particular an animal, in an effective antibiotic and/or anti-tumor and/or antiviral and/or antimicrobial and/or antispermicidal and/or antiparasitic amount, or in an amount effective to stimulate wound healing in a host. The peptides may be administered either alone or in combination with an ion having pharmacological properties, antibiotic, or ion channel-forming peptide or protein as hereinabove described. When the peptide is administered in combination with an ion having pharmacological properties, the activity of the peptide is potentiated.

When the peptide is administered in combination with an agent as hereinabove described, it is possible to administer the peptide and agent in separate forms. For example, the agent may be administered systemically and the peptide may be administered topically.

When the peptide is administered topically, it may be administered in combination with a water-soluble vehicle, said water-soluble vehicle being in the form of an ointment, cream, lotion, paste or the like. Examples of water-soluble vehicles which may be employed include, but are not limited to, glycols, such as polyethylene glycol, hydroxycellulose, and KY Jelly. The water-soluble vehicle is preferably free of an oily substance.

The peptide may also be employed alone, or in combination with other peptides of the present invention, or in combination with peptides containing L-amino acid residues, or in combination with other active components (eg., ions having pharmacological properties, antibiotics) as hereinabove described in the form of an oral composition for oral hygiene. Such a composition may be incorporated into a wide variety of compositions and materials used for oral hygiene purposes, which include, but are not limited to, toothpastes, mouthwashes, tooth gels, and tooth powders. Such composition may thus be used to treat or prevent periodontal disease, to prevent or reduce plaque, and/or to prevent or treat or reduce dental caries. The peptide may be used to inhibit, prevent, or destroy the growth of Streptococcus mutans, which is associated with dental caries and periodontal disease.

Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, within the scope of the accompanying claims, the invention may be practiced other than as particularly described.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 11                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        LysIleAlaGlyLysIleAlaLysIleAla                                                 510                                                                            GlyLysIleAlaLysIleAlaGlyLysIleAla                                              1520                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        GlyIleGlyLysPheLeuLysLysAlaLys                                                 510                                                                            LysPheGlyLysAlaPheValLysIleLeuLysLys                                           1520                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        GlyIleGlyLysPheLeuLysSerAlaLys                                                 510                                                                            LysPheGlyLysAlaPheValLysIleMetAsnSer                                           1520                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SER ID NO:4:                                        GlyIleGlyLysPheLeuLysLysAlaLys                                                 510                                                                            LysPheGlyLysAlaPheValLysIleMetLysLys                                           1520                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        LysLeuAlaSerLysAlaGlyLysIleAla                                                 510                                                                            GlyLysIleAlaLysValAlaLeuLysAlaLeu                                              1520                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        GlyIleGlyLysPheLeuLysSerAlaLys                                                 510                                                                            LysPheGlyLysAlaPheValLysIleLeuAsnSer                                           1520                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 22 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        GlyIleGlyLysPheLeuLysLysAlaLys                                                 510                                                                            LysPheAlaLysAlaPheValLysIleIleAsnAsn                                           1520                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 21 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (ix) FEATURE:                                                                  (D) OTHER INFORMATION:                                                         Xaa is p- aminophenylalanine.                                                  (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        LysIleAlaGlyXaaIleAlaLysIleAla                                                 510                                                                            GlyXaaIleAlaLysIleAlaGlyXaaIleAla                                              1520                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 12 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        LeuLeuLysLysLeuLysLysLeuLeuLysLysLeu                                           510                                                                            (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 14 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       LysLysLeuLeuLysLysLeuLysLysLeuLeuLysLysLeu                                     510                                                                            (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 11 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS:                                                              (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: peptide                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       LeuLysLysLeuLeuLysLysLeuLysLysLeu                                              510                                                                            __________________________________________________________________________ 

What is claimed is:
 1. A biologically active amphiphilic compound comprising:a peptide analog of Magainin I peptide or Magainin II peptide,said Magainin I peptide or Magainin II peptide analog being in an amide-or carboxy-terminated form, wherein Magainin I is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR35## wherein each amino acid residue which is not a glycine residue is a D-amino acid residue, and wherein Magainin II is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR36## wherein each amino acid residue which is not a glycine residue is a D-amino acid residue, and wherein said peptide analog is obtained by deleting the amino acid residue in position 19 and substituting at least one of amino acid residues 3, 7, 8, 10, 13, 15, 16, 18, 21, 22, or 23 with another amino acid as follows:

    ______________________________________                                         Residue Number   Substituent                                                   ______________________________________                                         3                D-Leu                                                         7                D-Lys                                                         8                D-Lys, D-Ala                                                  10               D-Ala, D-Lys                                                  13               D-Trp, D-Leu, D-Phe, D-Ala                                    15               D-Phe                                                         16               D-Ala                                                         18               D-Lys, D-Ala, D-Phe                                           21               D-Lys, D-Ile, D-Leu                                           22               D-Lys                                                         23               D-Lys, D-Ser, D-Asn.                                          ______________________________________                                    


2. The compound of claim 1, wherein the analog is an analog of Magainin II selected from the group consisting of:D-(SEQ ID No.2); D-(SEQ ID NO.3); D-(SEQ ID NO.4); D-(SEQ ID NO.6); and D-(SEQ ID No.7).
 3. A composition comprising the compound of claim 1 and a carrier.
 4. A biologically active amphiphilic peptidewhich is a peptide analog of Magainin I peptide or Magainin II peptide,said Magainin I peptide or Magainin II peptide analog being in an amide-or carboxy-terminated form, and wherein Magainin I is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR37## wherein each amino acid residue which is not a glycine residue is a D-amino acid residue, and wherein Magainin II is represented by the following structural formula using the single letter amino acid code and the numbers below each amino acid residue refer to the position of the residue in the peptide: ##STR38## wherein each amino acid residue which is not a glycine residue is a D-amino acid residue, and wherein said peptide analog is obtained by deleting the amino acid residue in position 19 and substituting at least one of amino acid residues 3, 7, 8, 10, 13, 15, 16, 18, 21, 22, or 23 with another amino acid as follows:

    ______________________________________                                         Residue Number   Substituent                                                   ______________________________________                                         3                D-Leu                                                         7                D-Lys                                                         8                D-Lys, D-Ala                                                  10               D-Ala, D-Lys                                                  13               D-Trp, D-Leu, D-Phe, D-Ala                                    15               D-Phe                                                         16               D-Ala                                                         18               D-Lys, D-Ala, D-Phe                                           21               D-Lys, D-Ile, D-Leu                                           22               D-Lys                                                         23               D-Lys, D-Ser, D-Asn.                                          ______________________________________                                    


5. A composition comprising the compound of claim 4 and a carrier.
 6. A pharmaceutical composition comprising the compound of claim 4 and an acceptable pharmaceutical carrier.
 7. A process for treating a microbial infection in a host, comprising the step of administering to the host an antimicrobially effective amount of the peptide of claim
 4. 8. A process for treating a tumor in a mammalian host, comprising the step of administering to the host an antitumor effective amount of the peptide of claim
 4. 